Genes encoding resistance to DNA alkylating agents

ABSTRACT

The invention provides genes encoding resistance to DNA bioreductive alkylating or cleaving agents and methods of identifying and using those genes.

This application is a continued provision of a national stage filing ofinternational application PCT/US94/11279, filed Oct. 6, 1994, which is acontinuation-in-part application of U.S. application Ser. No.08/133,963, filed Oct. 7, 1993, now abandoned.

BACKGROUND OF THE INVENTION

Agents that act to damage DNA by alkylation, DNA-DNA crosslinking,DNA-protein crosslinking, and/or DNA cleavage have been used as potentchemotherapeutic agents. Some of these agents are reductively activatedby electron transfer to a moiety such as semi-quinone. Agents which arethought to be reductively activated include the naturally occurring andsynthetic mitomycins and the enediynes such as neocarzinostatin. Kasaiet al., SynLett, 10:788 (1992) (mitomycins); and Nicolaou et al.,Angewandte Chemie, 30:1387 (1991) (enediynes). Cellular resistance tothese types of chemotherapeutic agents develop, especially in tumorcells. The mechanism of resistance of tumor cells and the organisms thatproduce the agents are not known.

Mitomycins are agents that are reductively activated and catalyze DNAalkylation and DNA-DNA crosslinks. Mitomycins are antitumor antibioticsproduced by Streptomyces lavendulae and other Streptomyces species.Several mitomycins have been characterized including the naturallyoccurring mitomycin A, mitomycin B, mitomycin C (MMC), porfiromycin, andmitiromycin. The structural properties and biological activity ofmitomycins have motivated a large number of studies to determinecellular target sites and mechanism of action. Complete structuralcharacterization of MMC has been followed by studies showing the basisof its remarkable activity against mammalian tumor cells. Iyer et al.,Science, 145:55 (1964); Schwartz et al., Science, 142:1181 (1963). Themolecule has three important functional groups, which include a quinonering system, carbamate moiety, and a highly strained aziridine groupthat contribute together to determine MMC target site specificity andability to alkylate DNA. Significantly, the precise regions in DNA thatundergo mono- and bifunctional alkylation by MMC, leading to inhibitionof replication and subsequent cell death have also been determined. Ceraet al., Biochemistry, 28:3908 (1989); Kumar et al., Biochemistry,32:1364 (1993); Teng et al., Biochemistry, 28:3901 (1989); and Tomasz etal., Science, 235:1204 (1987).

Mitomycins are DNA bioreductive alkylating agents which present adifficult and unique challenge for cellular resistance in producingmicroorganisms. Beijnen et al., J. Pharm. Biochem. Anal., 4:275 (1986);Kumar et al., supra. Evidence strongly supports the idea that the firststep in biological activation of mitomycins occurs by catalytic electrontransfer to the benza-quinone species to form a semi-quinone radicalshared by mitomycins. Hoey et al., Biochemistry, 27:2608 (1988). In thisform, the molecule would fit into the minor groove of DNA and undergofurther reduction to the hydroquinone. Beijnen et al., supra.; Hoey etal., supra. The second electron transfer is presumed to occur with rapidkinetics, and is followed by DNA alkylation through activation of thetwo electrophilic centers in the mitomycin hydroquinone species. Withthis unique biological activity, it is clear that the two most commonresistance mechanisms in bacteria, protection of the target site(methylation of ribosomal RNA) and modification of the antibiotic(phosphorylation or acetylation), are unlikely to be applicable tomitomycin. Davies, FEMS Microbiology Reviews, 39:363 (1986); Witt etal., Appl. Microbiol., 13:361 (1990). Specifically, it would beimpractical for the cellular DNA to be protected (e.g. modified byextensive methylation) because the GC content in S. lavendulae is ˜70%,and the mitomycin target site for mitomycin C (MMC) has been shownspecifically to be CpG residues. Teng et al., supra. Likewise,protection by drug modification through phosphorylation or acetylationwould not prevent electron transfer or the activation of electrophiliccenters in the molecule.

Although there is a wealth of knowledge about the structure, mode ofaction and mechanism of activation of mitomycin C, no studies have beenconducted to determine the molecular basis for resistance in theproducing organisms such as S. lavendulae. Indeed, resistance mechanismsare unknown for the entire class of bioreductive alkylating and cleavingagents like MMC, and other potent anti-tumor compounds that alkylate orcleave DNA following reductive activation. Beijnen et al., supra.; Hoeyet al., supra.; and Woo et al., J. Amer. Chem. Soc., 115:1199 (1993).Understanding resistance to these molecules in these producingmicroorganisms may provide insight into the problem of multidrugresistance (MDR) of cancer cells, and its effect on long termtherapeutic efficacy of antineoplastic agents. Moscow et al., MultidrugResistance. Cancer Chemotherapy and Biological Response Modifiers Annual11, Elsevier Science Publishers B.V. (1990). Identification ofadditional mechanisms that contribute to broad spectrum drug resistanceof tumors in mammalian systems may allow the development of strategiesto identify and effectively control this complex problem.

Thus, there is a need to study cellular resistance to compounds thatbioreductively alkylate or cleave DNA. There is a need to identifyagents that inhibit resistance to compounds that bioreductively alkylateor cleave DNA. There is also a need to identify and modify DNA genesequences that are responsible for drug resistance mechanisms inmicroorganisms and in animal and human tumor cells.

SUMMARY OF THE INVENTION

The invention provides for an expression cassette and vectors includingthe expression cassette. The expression cassette comprises a DNAsequence that provides resistance to a cell to a DNA bioreductivealkylating or cleaving agent operably linked to a promoter functional inthe cell. The preferred DNA bioreductive alkylating or cleaving agent isa mitomycin. Preferred DNA sequences are those that substantiallycorrespond to the mcr and mrd loci of S. lavendulae B619. The promoteris preferably functional in Streptomyces and provides for a sufficientlevel of gene expression so that resistance of the cell to the DNAbioreductive alkylating or cleaving agent can be detected.

Once a DNA sequence that provides resistance to a cell to a DNAbioreductive alkylating or cleaving agent is identified, it can be usedto generate DNA probes. A DNA probe has sufficient complementarity toall or a portion of a known DNA or RNA sequence that provides resistanceto the agent so that it can hybridize to the DNA or RNA sequence,preferably under low stringency conditions. Portions of a DNA or RNAsequence preferably are restriction endonuclease fragments of the mcr ormrd DNA sequence. The preferred probes are complementary to the 6.7 kbBclI fragment of pDHS3000 encoding mcr and the 4.2 kb BclI fragment ofpDHS3001 encoding mrd.

Two loci have been identified that provide mitomycin resistance tomitomycin sensitive host cells. One locus found on a 6.2 kb BclIfragment from S. lavendulae has now been designated mcr and is the sameas the locus designated mcrA in U.S. application Ser. No. 08/133,963. Onthe mcr locus, three open reading frames were identified and are nowdesignated 1) mcrA which is the same as the DNA sequence identified asmcrA1; 2) mcrB which is the same as the DNA sequence identified asmcrA2; and 3) mcrORF3 which is the same as the DNA sequence previouslyidentified as mcrAORF3 in U.S. application Ser. No. 08/133,963. Theother locus is found on a 4.2 kb BclI fragment on plasmid pDHS3001 andis now referred to as mrd and is the same as the locus previouslyidentified as mcrB in U.S. application Ser. No. 08/133,963. Theidentifiers of the gene loci and DNA sequences in this application havebeen changed from the parent application Ser. No. 08/133,963 asdescribed above. Subject matter from the parent application thatreferred to the previous identifiers has been modified to the newidentifiers, but the gene loci and DNA sequences remain the same asthose disclosed in the parent application Ser. No. 08/133,963.

The invention also provides for polypeptides and antibodies specific forthe polypeptides. A polypeptide can be encoded by a DNA sequence thatprovides resistance to a cell to a DNA bioreductive alkylating orcleaving agent such as the mcr DNA sequence. The preferred polypeptideis the MCRA polypeptide which is about a 56,000 dalton polypeptideencoded by mcrA.

The invention also provides transformed cells. Transformed cellscomprise an expression cassette comprising a DNA sequence thatsubstantially corresponds to a DNA sequence that provides resistance tothe cell to a DNA bioreductive alkylating or cleaving agent operablylinked to a promoter. The preferred cell is a cell sensitive to the DNAbioreductive alkylating or cleaving agent such as Streptomyces lividans.The preferred DNA sequence substantially corresponds to the DNA sequenceof the mcrA and mcrB genes. The expression cassette preferably isexpressed in an amount sufficient to confer resistance to the cell tothe agent.

The invention also provides methods for identifying agents that inhibitthe resistance of the cell to the DNA bioreductive alkylating orcleaving agent. One method involves using transformed cells. Transformedcells resistant to the agent comprise an expression cassette asdescribed herein. The transformed cells are incubated with an effectiveamount of an agent suspected to inhibit resistance of the cell to theDNA bioreductive alkylating or cleaving agent and an effective amount ofthe DNA bioreductive alkylating or cleaving agent. After incubation fora suitable amount of time, it can be determined if the suspected agentinhibited the resistance of the cell to the DNA bioreductive alkylatingor cleaving agent.

In an alternative version, an inhibitory agent can be identified by itsability to inhibit the function of a polypeptide encoded by the DNAsequence. A substantially pure polypeptide such as MCRA is incubatedwith a DNA sample and the DNA bioreductive alkylating or cleaving agentand the inhibitory agent. After incubation, it can be determined whetherthe inhibitory agent inhibited the function of MCRA polypeptide bymeasuring the binding of the DNA bioreductive alkylating or cleavingagent to the DNA sample or by determining whether DNA alkylation hasoccurred. If the suspected inhibitory agent inhibits the function ofMCRA, binding and/or activity of the DNA bioreductive alkylating orcleaving agent is increased in the presence of the suspected inhibitoryagent, preferably 2 to 20-fold.

The invention also provides a method for identifying sequenceshomologous to the mcr or mrd sequences in other cell types and/ororganisms. Preferably, the cells are multi-drug resistant or mitomycinC-resistant tumor cells. The method involves generating a DNA libraryand amplifying selected sequences in the library using polymerase chainreaction. Amplification of selected sequences is accomplished byselecting oligonucleotide primers that are complementary to a portion ofthe mcr or mrd loci. Once formed, the amplified products are isolatedand screened for homology to mcr or mrd by hybridization to a DNA probe.Sequences that hybridize can be mapped using restriction enzymes andsequenced using standard methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Model of the mechanism whereby the MCRA polypeptide confersresistance against MMC. The left hand side of the figure demonstrates anactivation cascade for MMC while the right side shows how MCRA canprevent MMC activation. After reduction of the quinone of MMC to thesemi-quinone radical, an FAD bound to MCRA would remove a singleelectron to form FADH·. FADH· could again be reduced completely to FADH₂or undergo reoxidation to FAD.

FIG. 2. Nucleotide sequence and deduced amino acid sequence of mcrA [SEQID NO: 1], mcrB [SEQ ID NO: 2], and mcrORF3 [SEQ ID NO: 3] genes of S.lavendulae B619. Shaded circles with arrows represent the translationalstart point (TSP) for promoters P1 and P2 as determined by primerextension. The thin arrowed line labeled PE primer indicates thelocation of the oligonucleotides used for TSP determination. Potential−35 and −10 regions are labeled for both TSPs. The dark line labeled RBSindicates a potential RBS for a polypeptide translated from the shortertranscript originating from P2. The shaded valine proximal to the RBSindicates a potential start codon for the shortened mcrA polypeptide.Facing arrows reveal two divergent repeats, which may have roles intranscription termination, with ΔG values lower than −45 kcal/mol.

FIG. 3. Alignment of the mcrA (PI) [SEQ ID NO: 5] and6-hydroxy-D-nicotine oxidase [SEQ ID NO: 4] deduced amino acidsequences. Identical amino acids are shaded. The histidine responsiblefor attachment of FAD to 6-hydroxy-D-nicotine oxidase and conserved inthe deduced mcrA amino acid sequence is marked by a dot (·).

FIG. 4. Dot plot comparisons of the mcrA deduced protein sequence with6-hydroxy-D-nicotine oxidase (6HDNO) and L-gulono-γ-lactone oxidase. (A)mcrA (horizontal axis) and 6-hydroxy-D-nicotine oxidase; (B) mcrA(horizontal axis) and L-gulono-γ-lactone oxidase. Analysis was performedusing a stringency of 33% identity with the range set at 10 amino acids.

FIG. 5A: Streptomyces lavendulae strains which produce MMC, probed witha 6.7 kb BclI insert of pDHS3000 encoding mcr. Lanes: 1, 1 kb ladder; 2,BclI S. lividans 1326; 3, BamHI S. lividans 1326; 4, BclI S. lavendulaeB619; 5, BamHI S. lavendulae B619; 6, BclI S. lavendulae PB1000; 7,BamHI S. lavendulae PB1000; 8, BclI S. lavendulae NRRL 2564; 9, BamHI S.lavendulae NRRL 2564; 10, BclI S. lavendulae KY681; 11, BamHI S.lavendulae KY681; and 12, λHindIII DNA ladder.

FIG. 5B: S. lavendulae probed with a 4.2 kb BclI insert of pDHS3001encoding mrd. Lanes: 1, 1 kb ladder; 2, BclI S. lividans 1326; 3, BamHIS. lividans 1326; 4, BclI S. lavendulae B619; 5, BamHI S. lavendulaeB619; 6, BclI S. lavendulae PB1000; 7, BamHI S. lavendulae PB1000; 8,BclI S. lavendulae NRRL 2564; 9, BamHI S. lavendulae NRRL 2564; 10, BclIS. lavendulae KY681; 11, BamHI S. lavendulae KY681; 12, λHindIII DNAladder.

FIG. 6. Frame analysis of mcr, and restriction enzyme map of pDHS3000illustrating subcloning performed to determine the DNA sequencenecessary to confer MMC resistance to S. lividans. A (+) in themitomycin C resistance column indicates that the vector to the right wasable to confer MMC resistance, whilst a (−) indicates no resistance.Shaded areas represent DNA cloned from S. lavendulae B619, whereas darkareas represent vector DNA.

FIG. 7. Restriction-enzyme map of the pPRA112 cosmid clone containing S.lavendulae DNA adjacent to the mcr locus.

FIG. 8. Restriction enzyme maps of the cosmid clones containing S.lavendulae DNA adjacent to the mrd locus. The central shaded regiondenotes the boundaries of the mrd locus.

FIG. 9. Strategy for gene-disruption by homologous recombination. Thethiostrepton resistance gene (tsr) was used for selection by cloninginto the middle of mcrORF3. Single-stranded DNA was then used totransform S. lavendulae, followed by selection with thiostrepton toobtain recombinant organisms.

FIG. 10. SDS-PAGE of purified MCRA. MCRA was purified as described inExample 4.

FIG. 11. DNA sequence of mrd locus derived from 4.2 kb BclI fragmentfrom plasmid pDHS3001 [SEQ ID NO: 11].

FIG. 12. Genetic map of mrd resistance locus from Streptomyceslavendulae. Shaded regions are defined as indicated. A putative MMChydroxylase is located at the 3′ end of mrd locus. Map of subclones ofmrd locus and the ability of the subclones to confer resistance to MMCat 25 μg/ml to S. lividans.

FIG. 13. MMC induction of MCRA expression in S. lividans/pDHS3000. PanelA shows Commassie blue stained SDS-PAGE gel of extracts from S.lividans/pDHS3000 grown with varying concentrations of MMC. Lane 1,purified MCRA control; Lane 2, S. lividans/pIJ702 grown with 1 μg/mlMMC; 3, S. lividans/pIJ702 grown with 0 μg/ml MMC; 4, S.lividans/pDHS3000 grown with 0 μg/ml MMC; 5, S. lividans/pDHS3000 grownwith 1×10⁻⁵ μg/ml MMC; 6, S. lividans/pDHS3000 grown with 1×10⁻⁴ μg/mlMMC; 7, S. lividans/pDHS3000 grown with 1×10⁻³ μg/ml MMC; 8, S.lividans/pDHS3000 grown with 1×10⁻² μg/ml MMC; 9, S. lividans/pDHS3000grown with 1×10⁻¹ μg/ml MMC; 10, S. lividans/pDHS3000 grown with 1 μg/mlMMC; 11, S. lividans/pDHS3000 grown with 5 μg/ml MMC; 12, S.lividans/pDHS3000 grown with 10 μg/ml MMC; 13, S. lividans/pDHS3000grown with 50 μg/ml MMC; Panel B shows Western blot of SDS-PAGE usinganti-MCRA antibodies; Panel C shows MCRA expression as a function of MMCconcentration.

FIG. 14. Mitomycin induction of MCRA expression in S. lividans/pDHS3000. represents cells incubated with 3 μm (1 μg/ml) of the mitomycins. □represents cells incubated with 15 μM of the mitomycins. MA=mitomycin A;MB=mitomycin B; MC=mitomycin C; MD=mitomycin D; MF=mitomycin F;MH=mitomycin H; Por=porfiromycin; none control; 702=p1J702.

FIG. 15. Induction of MCRA expression as measured by ELISA assay in S.lividans cells containing pDHS3000. Cells were treated with 76 mMneocarzinostatin (NCS), mephalan, (±) −1,2:3,4 diepoxybutane (DEB),daunomycin and mitomycin C.

FIG. 16. Panel A UV spectrum of purified MCRA. Panel B=amplifiedspectrum from panel A between 300-500 nm.

FIG. 17. 15 L fermentation of S. lavendulae showing MMC production, drymass and MCRA expression over the course of a 240 hr culture.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for an expression cassette including a DNAsequence that provides resistance to a cell to a DNA bioreductivealkylating or cleaving agent as well as a polypeptide encoded by the DNAsequence. The invention also provides transformed cell lines and methodsfor identifying agents that inhibit the resistance of a cell to a DNAbioreductive alkylating or cleaving agents. DNA probes of the inventionare substantially complementary to all or a portion of a DNA sequencethat provides resistance to a cell to a DNA bioreductive alkylating orcleaving agent and are useful in a method for detecting homologous DNAsequences in other organisms.

DNA sequences providing for resistance to a cell to a DNA bioreductivealkylating or cleaving agent have been identified. A DNA sequence thatprovides resistance to a DNA bioreductive alkylating or cleaving agentcan encode a polypeptide that imparts resistance to cells from theseagents and/or protects DNA from these agents.

A bioreductive DNA alkylating or cleaving agent is an agent that has amoiety that is reductively activated by electron transfer and cancatalyze cleavage or alkylation of DNA. Agents that have a moiety suchas a benza-quinone which is reduced to a semiquinone and/or hydroquinonespecies to become bioreductively activated include mitomycins. While notmeant to be a limitation of the invention, it is believed that once theDNA bioreductive alkylating or cleaving agent is reductively activatedit binds to DNA and can catalyze one or more of the following reactions:DNA alkylation, DNA-DNA crosslinking, DNA-protein crosslinking, or DNAcleavage. It is further believed that one way that a polypeptide impartsresistance to cells from these agents is by binding to and deactivatingthe reduced active form of the agent. It is believed that deactivationoccurs by oxidation.

A specific example of one cellular resistance mechanism to DNAbioreductive alkylating or cleaving agents is shown in FIG. 1. The DNAbioreductive alkylating or cleaving agent shown in FIG. 1 is mitomycin Cand a polypeptide that imparts resistance to mitomycin C (MMC) isdesignated MCRA. While not meant to be construed as a limitation of theinvention, it is believed that the MCRA polypeptide includes acovalently bound cofactor, such as flavin adenine dinucleotide (FAD),that mediates the oxidation of MMC in the event that it undergoessequential reduction through the semi-quinone radical. One electronreduction of MMC can result in immediate oxidation by MCRA bound FAD tothe FADH· species. The FADH· species would be available to accommodate asecond electron transfer from MMC to result in a non-reduced form of MMCthat cannot bind to or catalyze alkylation or crosslinking of DNA.

Identification of DNA sequences that provide resistance to a cell to aDNA bioreductive alkylating or cleaving agent provides for polypeptides,DNA probes, and transformed cell lines. These DNA sequences can be usedin methods to identify agents that inhibit resistance of cells to a DNAbioreductive alkylating or cleaving agent as well as to identifyhomologous DNA sequences that provide for resistance in other cells,such as tumor cells.

The invention also provides for antibodies to MCRA and methods fordetecting expression of MCRA or related polypeptides in cells resistantto DNA bioreductive alkylating and/or cleaving agents.

A. Bioreductive Alkylating or Cleaving Agents

Agents that act to damage DNA by alkylation, DNA-DNA crosslinking,DNA-protein crosslinking, and/or DNA cleavage can be potent cancerchemotherapeutic agents. These agents are characterized by the abilityto bind to and alter DNA. A DNA cleaving agent is one that binds DNA andcreates single or double-stranded breaks. A DNA alkylating agent is anagent that binds to DNA and forms covalent bonds to the DNA molecule.DNA alkylation includes the formation of DNA-DNA and DNA-proteincrosslinks.

Within this class of compounds are compounds referred to as DNAbioreductive alkylating or cleaving agents. A DNA bioreductivealkylating or cleaving agent is a compound that is reductivelyactivated. Reductive activation can occur by electron transfer to amoiety in the agent such as a benza-quinone. Molecules containingmoieties such as anthraquinone, aminoquinone, or enediyne can becomereductively activated and then catalyze damage to DNA. Compounds thatcan be reductively activated preferably include the following structuralcomponent:

Compounds that include both an aromatic ring as shown above andoptionally include an aziridine nitrogen group at carbons 1 and 2 arealso perferred DNA bioreductive alkylating or cleaving agents. While notmeant to be a limitation of the invention, it is believed that once theagent is activated, it can bind to DNA and catalyze DNA alkylation,crosslinking or cleavage. An aromatic group can function as a site forreductive activation and an aziridine group is involved in creating DNAdamage.

The DNA bioreductive alkylating and/or cleaving agents are alsopreferably those compounds that can induce expression of a DNA sequencethat codes for resistance to the agent. A DNA bioreductive alkylating orcleaving agent can also be a compound that does not induce expression ofa DNA sequence that encodes resistance but that is inactivated by apolypeptide encoded by a DNA sequence that provide resistance to a cellto the agent. While not meant to limit the invention, it is believedthat inactivation of the DNA bioreductive alkylating or cleaving agentby a polypeptide occurs by binding of the agent to the polypeptidefollowed by deactivation by oxidation of the DNA bioreductive alkylatingor cleaving agent.

Specific example of bioreductive DNA alkylating or cleavage agentsinclude the mitomycins and related compounds. The structure ofmitomycins is as follows:

Mitomycins contain a benza-quinone moiety that can be reduced byelectron transfer to form activated mitomycins. Activated mitomycinsbind to DNA and catalyze DNA-DNA and DNA-protein crosslinks and DNAalkylation.

Mitomycins can be naturally occurring compounds produced by Streptomycesspecies and include mitomycin A, mitomycin B, mitomycin C, porfiromycin,and sit mitiromycin. Other mitomycins include mitomycins E, G. I, J, L,M, K, mitiromycin, albomitomycin A, A isomitomycin A, KW 2149, KW-2149metabolites such as M-16 and M-18. Naturally occurring mitomycins can beisolated from growth media of Streptomyces species by known methods, asdescribed Herr et al., Antimicrobial Agents Annual, at page 23, PlenumPress, NY (1960). Other Mitomycin derivatives have also been chemicallysynthesized as described Kasai et al. in SynLett, 10:777 (1992).Mitomycin analogs can also be obtained by directed biosynthesis asdescribed by Claridge et al., J. Antibiotics, 39:437 (1986).

Another specific example of DNA bioreductive alkylating or cleavingagents are substituted dihydrobenzoxazines such as FR900482, asdescribed in J. Am. Chem. Soc., 109:4106 (1987). The structure ofFR900482 is as follows:

These compounds are known to catalyze DNA-DNA and DNA-protein crosslinkssimilar to mitomycin C. Masuda et al., Cancer Research, 48:5172, citedsupra. The agent FR900482 can be obtained from growth medium ofStreptomyces sandaensis 6897, as described by Kiyoto et al., J.Antibiotics, 40:594 (1987).

DNA bioreductive alkylating or cleaving agents can be produced byStreptomyces species. Because of their toxic and damaging effects onDNA, the producing microorganisms have resistance mechanisms thatprotect their DNA from these agents. DNA sequences that impartresistance to DNA bioreductive alkylating or cleaving agents areisolated as described herein.

B. Expression Cassettes Including a DNA Sequence that ProvidesResistance to a Cell to a DNA Bioreductive Alkylating or Cleaving Agent

The invention provides an expression cassette comprising a DNA sequencethat provides resistance to a cell to a DNA bioreductive alkylating orcleaving agent operably linked to a promoter functional in the cell. TheDNA sequence can contain one or more genes and/or portions of the genesor gene locus. The DNA sequence can encode a single polypeptide or morethan one polypeptide. The DNA sequence can provide resistance to a DNAbioreductive alkylating or cleaving agent by encoding a polypeptide thatprovides resistance to the cell or protects DNA from the action of theDNA bioreductive alkylating or cleaving agent. The promoter ispreferably a DNA sequence that provides for a sufficient level ofexpression of the gene or genes encoded on the DNA sequence in the cellso that resistance of the cell to the DNA bioreductive alkylating orcleavage agent can be detected.

The DNA sequence can encode a gene locus. A “gene locus”, as usedherein, encodes more than one gene or has more than one open readingframe. A “gene”, as used herein, includes the DNA coding sequence andany intron DNA sequences. Untranslated sequences, such as promoters orenhancer sequences or 3′ polyA sequences or other untranslatedregulatory sequences that provide for gene expression in a cell, are notincluded in the term “gene” as used herein. A DNA sequence can alsoinclude portions of a gene locus or a gene. Portions of a gene or genelocus, as used herein, refer to restriction enzyme fragments of the geneor gene locus.

A cell is resistant to a DNA bioreductive alkylating or cleaving agentif it can grow in the presence of amounts of the agent that typicallyprevent growth of this type of cell. Growth preferably is about 2 to1000-fold, and more preferably 2 to 100-fold increased over a sensitivecell. Those amounts can vary depending on the cell and the agent, butcan be readily determined using standard dose response methods asdescribed in Masuda et al., cited supra. Preferably the amount of DNAbioreductive alkylating or cleavage agent is in the range of about1-1000 μg/ml, more preferably about 10-500 μg/ml, and most preferablyabout 25-100 μg/ml.

Alternatively, a cell is resistant to a DNA bioreductive alkylating orcleavage agent if the DNA from the cell does not develop interstrandDNA-DNA crosslinks or breaks in the DNA in the presence of the agent.DNA-DNA crosslinks can be determined by standard methods, such as thealkaline elution method or DNA renaturation method as described byMasuda et al., cited supra. Single strand DNA breaks in cells incubatedin the presence of a DNA bioreductive alkylating or cleaving agent canbe determined using a method such as the alkaline elution methoddescribed by Masuda et al., cited supra. Cells exhibiting resistance donot develop DNA-DNA crosslinks or breaks to any appreciable extent afterexposure to amounts of the agent that cause DNA-DNA crosslinks or breaksin the DNA in sensitive cells. Preferably, resistant cells show adecrease in DNA-DNA crosslinks of about 2 to 20 fold compared tosensitive cells.

A DNA sequence that provides resistance to a DNA bioreductive alkylatingor cleaving agent can encode a polypeptide that provides resistance tothe cell or protects the DNA from the action of the agent. Thispolypeptide can act to prevent transport of the agent into the cell ornucleus or can act to inactivate the agent. While not in any way meantto be a limitation of the invention, a DNA sequence can also provideresistance to a cell by specifying a structural alteration of the agent,or modifying cellular target sites. Preferably the polypeptideinactivates the bioreductive DNA alkylating or cleaving agent byoxidation.

A DNA sequence that imparts resistance to a cell to a DNA bioreductivealkylating or cleaving agent can include one gene or more than one gene.Preferably, resistance is imparted by the mcrA and mcrB genes on the mcrlocus providing for the expression of MCRA.

A DNA sequence that confers resistance to a cell to DNA bioreductivealkylating or cleavage agents can be isolated from the genome of thecell as follows. Preferably, the cell is a microorganism that produces aDNA bioreductive alkylating or cleaving agent. A DNA library can begenerated from the cell using standard methods as described in Sambrooket al., cited supra. One such method includes digesting the totalchromosomal DNA with the restriction enzyme and ligating the fragmentsinto a vector, such as a plasmid. The vectors are then introduced into ahost cell. The host cell is preferably a cell that does not grow in thepresence of the DNA bioreductive alkylating or cleaving agent (i.e., issensitive). Transformed cells are then selected for the ability to growin the presence of different amounts of the DNA bioreductive alkylatingor cleaving agent. An additional or alternative screening method thatcan be utilized optionally is to determine whether the transformed cellsthat can grow in the presence of the DNA bioreductive alkylating orcleaving agent develop DNA-DNA crosslinks or breaks as described herein.

Vectors from the transformed cells resistant to the agent are isolatedand the DNA sequences conferring resistance are identified. The abilityof the DNA sequences to impart resistance to the cell is confirmed bysubcloning of the sequence and transfer of the sequence into a sensitivehost cell. Analysis of the sequence of DNA is conducted by standardmethods. The DNA sequence and/or the predicted amino acid sequence canthen be compared to other known DNA sequences using a computer databank,such as the GenBank, and homologous sequences from other organismsidentified.

Portions of a gene or gene locus can be obtained by digesting a DNAsequence encoding the gene or gene locus with one or more restrictionenzymes such as shown in FIG. 6 and FIG. 12. The restriction enzymefragments of the DNA sequence are those fragments of the DNA sequencethat can confer resistance to a DNA bioreductive alkylating or cleavingagent to a cell. The fragments generated are ligated into a vector andthe vector is introduced into a suitable sensitive host cell. Thetransformed cells are selected for the growth in the presence of the DNAbioreductive alkylating or cleaving agent. Fragments of the gene or genelocus are identified by standard methods including restrictionendonuclease mapping.

Methods of transformation and suitable host cells are known to those ofskill in the art. Methods of transformation include calcium chloride orphosphate precipitation, electroporation, polybrene, liposomes, andprotoplast fusion with polyethylene glycol and the like. Suitable hostcells are those that exhibit sensitivity (i.e., do not grow in thepresence of the DNA bioreductive alkylating or cleaving agent). Specificexamples of suitable host cells are sensitive Streptomyces species suchas Streptomyces lividans, Streptomyces coelicoler, Streptomycesparvulus, and Streptomyces griseus.

Preferred examples of DNA sequences that provide resistance to a DNAbioreductive alkylating or cleaving agent include DNA sequences thatprovide resistance to mitomycins such as the mcr gene locus and the mrdgene locus from S. lavendulae B619. These two gene loci provide forresistance of S. lividans to mitomycin C. The mcr locus confersresistance to >100 μg/ml mitomycin C, and the mrd locus confersresistance to about 25 μg/ml. The mcr locus includes three differentcoding sequences designated mcrA, mcrB, and mcrORF3 and has the DNAsequence as shown in FIG. 2. The mcr locus is contained on the 6.7 kbBclI fragment of pDHS3000. The mcrA and mcrB sequences are found on the2.2 kb BglII-SphI fragment of pDHS3005.

The mrd locus is contained on the 4.2 kb BclI fragment of pDHS3001. Themrd locus is contained on the 4.2 kb BclI fragment of pDHS3001 and hasthe sequence shown in FIG. 11.

Preferred examples of portions of the DNA sequence are shown in FIG. 12.Subclones of the mrd locus are generated as follows. A 3280 bp subcloneof pDHS3001 is generated with AflIII/AscI. A 3152 bp subclone ofpDHS3001 was generated with NotI. These subclones confer resistance to acell to 25 ug/ml mitomycin C.

Preferred examples of portions of DNA sequences include those shown inFIG. 6. Portions of the mcr locus were generated by digesting a 6.7kilobase BclI fragment encoding the mcr locus with SphI, PuvII, BamHI,BglII, PstI, FspI, NcoI, StuI, and ClaI. The fragments are subclonedinto plasmids, as shown in Table I. The subclones are tested for theability to confer resistance to mitomycin C, as described herein. Theminimum DNA sequence of the mcr locus identified that providesresistance to mitomycin C corresponds to the DNA sequence of the 2.2kilobase BglII-SphI subclone of the plasmid designated pDHS3005.

DNA sequences can also substantially correspond to DNA sequencesencoding the mcr gene locus, mcrA, mcrB, and mcrORF3, respectively. ADNA sequence that substantially corresponds is a DNA sequence thatshares sufficient continuous DNA sequence identity to a DNA sequenceencoding the mcr locus, mcrA gene, mcrB gene, or mcrORF3 gene or mrdlocus so that the DNA sequence provides resistance to a cell tomitomycins, and can hybridize to a probe derived from mcr or mrd locus.A DNA sequence that substantially corresponds preferably shares about75-100% DNA sequence identity, and more preferably about 90-100% DNAsequence identity. Once the sequence is known as shown in FIGS. 2 and11, probes can readily be designed and synthesized using methods knowntwo those of skill in the art. The sequence preferably hybridizes to aprobe derived from the mcr or mrd loci under conditions of highstringency. One example of a DNA sequence that substantially correspondsto the DNA sequence of mcrA is a DNA that includes a CAT codon for His⁶⁴rather than CAC. Changes in the nucleotide sequence that do not resultin a change in the amino acids encoded by the DNA sequence are sequencesthat are likely to also provide resistance to a cell to mitomycins.

It would be understood by those of skill in the art that due to thedegeneracy of the genetic code, there is a defined set of DNA sequencesthat can encode polypeptides encoded by mcr and mrd loci such as MCRA.These DNA sequences can vary in the DNA sequence due to changes in acodon for a particular amino acid, but still encode a polypeptide withan amino acid sequence such as that of MCRA. These DNA sequences arealso those that impart resistance to a cell to a DNA bioreductivealkylating and/or cleaving agent.

Promoters

A DNA sequence that provides for resistance to a DNA bioreductivealkylating or cleaving agent to a cell functional in the cell in theexpression cassette is operably linked to a promoter. A promoter is anuntranslated DNA sequence that provides for expression of the DNAsequence and is preferably located immediately upstream from the DNAsequence. Preferably, the promoter provides for a level of expression ofthe DNA sequence in an amount effective to render the cell resistant tothe DNA bioreductive alkylating or cleaving agent. The promoter can be anative promoter that is associated with and provides for expression ofthe DNA sequence in the source organism. The promoter can be aconstitutive or inducible promoter. The promoter can also be aheterologous promoter obtained from a different gene and/or a differentorganism. The promoter is functional in the host cell carrying theexpression cassette. The promoter can be derived from prokaryotic,viral, or eukaryotic sources.

Suitable examples of prokaryotic promoters include the P_(Lac) promoter,the P_(tac) promoter, mel promoter on pIJ702, tipA of Streptomyceslividans and ermE of Saccharopolyspora erythrea. Suitable examples ofviral promoters include the SV40 early promoter, the Herpes Simplexthymidine kinase promoter, the Rous sarcoma LTR promoter, thebacteriophage T7 promoter, the bacteriophage λP_(L) promoter, and thelike. Suitable examples of eukaryotic promoters include yeast promoterssuch as ADHI promoter, the TPI promoter, the GALI promoter, and themetalothionein promoter. The preferred promoters for an expressioncassette are the P_(tac) promoter and the SV40 early promoter. Theespecially preferred promoter is a promoter inducible by the DNAbioreductive alkylating or cleaving agent. Promoters are commerciallyavailable in vectors or can be obtained using known methods as describedin Sambrook et al., cited supra., and Nielsen et al., Appl. Microbiol.Biotech., 33:307 (1990).

The promoter sequence is combined with a DNA sequence that providesresistance to a DNA bioreductive alkylating or cleaving agent bystandard methods to form an expression cassette. One such methodinvolves digesting a plasmid containing the DNA sequence and a plasmidcontaining the promoter with the same restriction enzymes, and thenligating the fragments into a third vector. The third vector is thentransformed into host cells and the host cells are selected forexpression of the DNA sequence by selecting cells resistant to the DNAbioreductive alkylating or cleaving agent. Other methods utilizingsubcloning of the DNA sequence into established expression vectors canbe conducted as described by Sambrook et al., cited supra, forexpression of cloned DNA sequences in mammalian or prokaryotic cells.

Once formed, an expression cassette can also be subcloned into a vectorso that efficient transmission into sensitive host cells can beachieved. Vectors can include viral or plasmid vectors. Preferably, thevector is a known expression vector such as the SV40 vectors, the pSMG,the pSVT7, the pMT2, the p205, the pHeBo, the pBV-1MTHA, the PAS1, thepET-3A, and the pKK177-3 as described by Sambrook et al., at pages 16.17to 16.27. The preferred expression vector is a high copy number plasmidfrom Streptomyces species such as the pIJ702. Many of the expressionvectors are commercially available. pIJ702 can be obtained from the JohnInnes Institute, Norwich, England.

An expression cassette can also comprise a selectable marker gene.Selectable marker genes are known to those of skill in the art and arepresent in the commercially available expression vectors. Specificexamples of selectable marker genes include thymidine kinase,dihydrofolate reductase, aminoglycoside phosphotransferase, hygromycin Bphosphotransferase, adenosine deaminase, arginine synthetase, andantibiotic resistance genes.

A preferred plasmid comprising an expression cassette in accordance withthe invention is the plasmid pDHS3000 with a 6.7 kb BclI DNA sequencefrom S. lavendulae B619 containing the mcr locus. This plasmid has beendesignated 1326/pDHS3000 and deposited in Streptomyces lividans with theAmerican Type Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) and given Accession No. 69448. Another preferred plasmid isthe pDHS3001 with a 4.2 kb BclI DNA sequence from S. lavendulae B619that contains the mrd locus. This plasmid has been designated1326/pDHS3001 and deposited in Streptomyces lividans with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) and given Accession No. 69449.

Once an expression cassette is formed, it can be used to formtransformed cells. The transformed cells can provide polypeptidesencoded by the DNA sequence and that provide resistance to DNAbioreductive alkylating or cleaving agents. The transformed cells arealso useful in methods for identifying agents that inhibit theresistance of cells to the DNA bioreductive alkylating or cleavingagents.

C. DNA Probes

Once a DNA sequence that provides resistance to a DNA bioreductivealkylating or cleaving agent is identified, the sequence can be used todevelop DNA probes. DNA probes can be useful in a method for identifyinghomologous DNA sequences that provide resistance to a DNA bioreductivealkylating or cleaving agent in other cells such as drug-resistant tumorcells.

A DNA probe in accordance with the invention comprises a DNA sequencethat has sufficient DNA sequence complementarity to all or a portion ofa known DNA or RNA sequence that provides for resistance to a DNAbioreductive alkylating or cleaving agent so that the probe can detectthe DNA or RNA sequence by hybridization under low stringencyconditions. The probe preferably hybridizes to all or a portion of mcror mrd locus under high stringency conditions. The DNA sequence can beas small as about 17 nucleotides and can be single or double stranded.Preferably, the DNA probe has a size of about 17-2500 nucleotides andmore preferably about 50-220 nucleotides.

A preferred DNA probe includes a sequence complementary to nucleotides310-330 of the mcrA sequence. This nucleotide sequence includes thecodon for the histidine residue (His⁶⁴) believed to be important forbinding of the FAD cofactor. Other preferred probes include DNAsequences complementary to the DNA sequence found at nucleotides 142 to166 for amino acids 171 to 177, and nucleotides 646 to 663 for aminoacids 429 to 436 of the mcrA gene. As shown in FIG. 3, these regionsshare amino acid homology with the 6-hydroxy-D-nicotine oxidase. Whilenot in any way meant to limit the invention, it is believed that thepolypeptides that provide resistance to mitomycins and other DNAbioreductive alkylating or cleaving agents have a similar mechanism ofaction as 6-hydroxy-D-nicotine oxidase. Region of the DNA sequence thatencode amino acids that share homology with the DNA sequence for the6-hydroxy-D-nicotine oxidase are selected to develop DNA probes.

Preferred DNA probes also include probes that are complementary to allor a portion of the DNA sequence for the mcr and mrd gene locus. Theseprobes include sequences complementary to the DNA sequence of 6.7 kbBclI fragment of pDHS3000 encoding the mcr locus and the 4.2 kb BclIfragment of pDHS3001 encoding the mrd locus. These probes have been usedto identify DNA sequences in the genome of resistant Streptomyces spp.by hybridization as shown in FIG. 5. Portions of the DNA sequence caninclude restriction enzyme fragments, preferably those shown in FIG. 6or FIG. 12.

DNA probes can be prepared by standard methods such as automated DNAsynthesis or as restriction endonuclease fragments. DNA probes arepreferably labelled with a detectable agent such as a radioactivelylabelled nucleotide. Once a sequence is selected, a DNA probe labelledwith the detectable agent can be prepared by automated DNA synthesis,polymerase chain reaction, nick translation, and other methods asdescribed in Sambrook et al.

DNA probes are used to detect homologous sequences by hybridization. ADNA probe according to the invention has sufficient complementarity to aDNA or RNA sequence so that it can hybridize to a DNA or RNA sequenceunder either low or high stringency conditions. Sufficientcomplementarity depends on the length of the probe, whether anymismatches are present on the probe, and the stringency conditions andthe effect of these factors on hybridization are known to those of skillin the art. Typically, in hybridizations conducted under lowerstringency conditions, the probe can be smaller in length and have somemismatches in sequences. Methods of DNA-DNA and DNA-RNA hybridization aswell as high and low conditions of stringency are known to those ofskill in the art and are described in Sambrook et al.

D. Transformed Cells

Once an expression cassette is formed in accordance with the invention,it can be used to form transformed cells. Transformed cells carrying aexpression cassette can be used in method to identify agents thatinhibit resistance of the cell to the DNA bioreductive alkylating orcleaving agent. Transformed cells include an expression cassettecomprising a DNA sequence that provides resistance to a cell to a DNAbioreductive alkylating or cleaving agent operably linked to a promoterfunctional in the cell. The preferred DNA sequence substantiallycorresponds to the DNA sequence for the mcrA and mcrB genes. Theexpression cassette provides the cell with resistance to the DNAbioreductive alkylating or cleaving agent.

A transformed cell can be formed by standard methods using an expressioncassette of the invention prepared as described herein. Briefly, apreferred expression cassette comprising a DNA sequence thatsubstantially corresponds to a DNA sequence of mcrA and mcrB genesoperably linked to a promoter is combined with a vector such as theplasmid pIJ702. A plasmid carrying the expression cassette is introducedinto a suitable host by transformation methods such as calcium phosphateor calcium chloride precipitation, liposomes, electroporation, and thelike. Transformants are selected for growth in the presence of the DNAbioreductive alkylating or cleaving agent. Transformed cells that areselected for growth in the presence of the agent are considered to beresistant to the agent.

Preferred host cells are those cells whose growth is inhibited in thepresence of the DNA bioreductive alkylating or cleaving agent in theabsence of the expression cassette (i.e., are sensitive). Specificexamples of sensitive cells include Streptomyces species such asStreptomyces lividans, tumor cells such as the L1210, the EMT6, HG-29,BE human carcinoma, and the L5178Y. The preferred host cell isStreptomyces lividans. Specific examples of a DNA bioreductivealkylating or cleaving agent include mitomycin A, mitomycin B, mitomycinC, profiromycin, mitiromycin, and FR900147. The preferred DNAbioreductive alkylating or cleaving agents are mitomycin compounds.

In a preferred version, the plasmid pDHS3005 is introduced intoprotoplasts of S. lividans by polyethylene glycol transformation.Plasmid pDHS3005 comprises the 2.2 kb BalII-SphI DNA sequence includingthe mcrA and mcrB gene sequences. S. lividans transformants are selectedfor growth in the presence of about 10 to 100 μg/ml of mitomycin C.Transformed cells that can grow in the presence of at least about 10μg/ml of mitomycin C are considered resistant to mitomycin C.

A transformed cell can also be a transformed cell line. A transformedcell line can be formed by amplifying the transformed cells selected forresistance to the agent. Methods of amplification and subculturing ofcells to form homogeneous cell lines are known to those of skill in theart. A preferred sensitive cell line is a tumor cell line sensitive tomitomycins such as the L1210 cell line. A transformed cell line canexhibit transient expression of resistance of about 48 to 72 hours orcan exhibit stable expression of resistance over several generations ofgrowth (i.e., about 50-100 generations) in the presence of the DNAbioreductive alkylating or cleaving agent.

E. Polypeptides that are Encoded by DNA Sequences that ProvideResistance to a Cell to a DNA Alkylating or Cleaving Agent andAntibodies Thereto

The invention also provides polypeptides that are encoded by DNAsequences that provide resistance to a cell to a DNA bioreductivealkylating or cleaving agent. While not meant to be a limitation of theinvention, polypeptides can provide resistance to the cell to a DNAbioreductive alkylating or cleaving agent by inhibiting transport of theagent, deactivating the agent, inhibiting binding of the agent to DNA,or inhibiting the crosslinking or cleaving of DNA by the agent.

A polypeptide encoded by the DNA sequence that provides resistance to acell to the agent can be identified by its presence in cell lysates oftransformed cells compared with its absence in non-transformed cellsusing standard methods. A polypeptide can also be identified bydetermining whether the polypeptide can inhibit the binding to oractivity of the DNA bioreductive alkylating or cleaving agent with a DNAsample by standard methods. Development of DNA-DNA crosslinks or breaksin the presence of the polypeptide and the agent can be assayed by thealkaline elution method or the DNA renaturation method as described byMasuda et al., cited supra. Once identified, polypeptides can beisolated from transformed cell lysates using standard methods. Preferredpolypeptides are encoded by the mcr locus can have a molecular weightwithin the range of about 10 to 60 kD as measured by SDS-PAGE.

An especially preferred example of a polypeptide that is encoded by aDNA sequence that provides resistance to a DNA alkylating or cleavingagent is the MCRA polypeptide. The MCRA polypeptide is about a 56,000dalton molecular weight polypeptide as determined by SDS-PAGE that canbe isolated from a transformed cell such as S. lividans carrying theplasmid pDHS3000. A portion of the N-terminal sequence of the MCRApolypeptide isolated from transformed cell lysates is identical to thatof the predicted amino acid sequence for the polypeptide encoded by themcrA gene as shown below:

Predicted MCRA Sequence: MSTQWGWALEPDQPGY (SEQ ID NO: 12)

N-terminal Sequence of isolated MCRA: STQWGWALEPD (SEQ ID NO: 13)

The predicted amino acid sequence of the MCRA polypeptide shares aminoacid homology with the 6-hydroxy-D-nicotine oxidase and L-gulono-lactoneoxidase. Both of these enzymes catalyze reactions using FADcofactor-mediated oxidation.

It is believed that a cofactor such as FAD binds to the His⁶⁴ residue ofMCRA and mediates oxidative deactivation of a DNA bioreductivealkylating or cleaving agent such as mitomycin compounds. The UVspectrum of purified MCRA and electrospray spectrometry indicates thatFAD is covalently bound to MCRA as a cofactor.

MCRA and other polypeptides that are encoded by DNA sequences thatimpart resistance to DNA bioreductive alkylating or cleaving agents canbe analyzed by inhibition of DNA or binding to alkylating or cleavingagents that are radiolabeled. This inhibition can be assessed by bindingof the polypeptide to a radiolabeled agent or by inhibition ofactivation of the agent in the presence of a reducing agent. Thepolypeptides preferably have oxidase activity and prevent reductiveactivation of the alkylating and/or cleaving agents.

The invention also includes antibodies to polypeptides encoded by DNAsequences that provide resistance to a cell to a DNA alkylating orcleaving agent. Once such polypeptides are identified, antibodiesspecific for the polypeptides can be prepared by standard methods knownto those of skill in the art.

The antibodies can be used in assays to detect expression ofpolypeptides, to isolate and purify polypeptides and DNA sequencesencoding them, and to identify and isolate related polypeptides in otherresistant cells.

Polyclonal antibodies can be formed by injecting an animal such as amouse several times intravenously with a polypeptide such as MCRApolypeptide. Typically about 0.5 to 2.0 mg are injected with Freund'sincomplete adjuvant on at least three separate occasions. About one totwo weeks after the last immunization, sera can be collected and theantibodies to a polypeptide such as MCRA can be quantitated using astandard ELISA test.

Monoclonal antibodies can be formed using the standard Kohler, Milsteintechnique. Pursuant to the Kohler, Milstein technique, immunization ofthe mammalian host is accomplished within the dose parameter bysubcutaneous or intraperitoneal injection of the immunogen compound inadjuvant. Administration is repeated periodically and preferably for atleast three injections. Three days before the spleen is removed, apriming injection of the immunogen compound is again administered. Aftertheir separation, the spleens are fused with the immortal mammaliancells such as mouse myeloma cells using the techniques outlined byKohler and Milstein. Polyethylene glycol (PEG) or electrical stimulationwill initiate the fusions. The fused cells are then cultured in cellwells according to culture techniques known in the art. Cellularsecretions in the culture medium are tested after an appropriate timefor the presence of the desired monoclonal antibodies.

The selection technique for identifying the appropriate monoclonalantibodies is an important aspect for determining the immunospecificityof the monoclonal antibody. The selection techniques call fordetermining the binding affinity of the hybridoma cellular products forpolypeptides such as the MCRA and against cross-reactive controls. Inparticular, hybridoma culture fluid is tested in screening assays forimmunoreactivity with the polypeptide MCRA and lack of immunoreactivitywith bovine serum albumin.

Screening assays can be performed by immunoenzymatic assay,immunofluorescence, fluorescenceactivated cell sorter, radioimmunoassay,immunoprecipitative assay or inhibition of biological activity. Thehybridoma culture selected will exhibit strong binding characteristicsto a polypeptide such as the MCRA polypeptide and exclude binding with avariety of controls including bovine serum albumin and a polypeptideencoded with the mcrB gene.

Following the identification of cell cultures producing the desiredmonoclonal antibodies, subcloning to refine the selected culture can beperformed. These techniques are known to those skilled in the art. See,for example, Goding, James Goding, Monoclonal Antibodies: Principles andPractice, 2nd Edition, academic Press, San Diego, Calif. (1986).

F. Methods for Identifying Agents that Inhibit Resistance of a Cell to aDNA Bioreductive Alkylating or Cleaving Agent

The invention also provides for methods of identifying agents thatinhibit the resistance of cells to a DNA bioreductive alkylating orcleaving agent. Applications of such methods include identification ofdrugs or agents that might be useful to combat development of tumor drugresistance and design of analogs of the DNA bioreductive alkylating orcleaving agents that resistant cells are sensitive to.

One method of identifying agents that inhibit resistance of cells to aDNA bioreductive alkylating or cleaving agent involves the use of atransformed cell. A step of the method involves providing a transformedcell that is resistant to the DNA bioreductive alkylating or cleavingagent. The transformed cell comprises an expression cassette having aDNA sequence that provides resistance to a cell to a DNA bioreductivealkylating or cleaving agent, preferably the DNA sequence thatsubstantially corresponds to the DNA sequence for the mcrA and mcrBgenes, operably linked to a promoter functional in the cell. Thetransformed cell is incubated with an effective amount of an agentsuspected to inhibit resistance of the cell to the DNA bioreductivealkylating or cleaving agent and an effective amount of the DNAbioreductive alkylating or cleaving agent. After suitable incubation, itcan be determined whether the suspected agent inhibited the resistanceof the cell to the DNA bioreductive alkylating or cleaving agent.

The transformed cell can be obtained as described herein. A preferredtransformed cell is S. lividans carrying the pDHS3005 plasmid encodingthe mcrA and mcrB genes. The DNA bioreductive alkylating or cleavingagent is preferably a mitomycin and the especially preferred agent ismitomycin C.

The agent suspected to inhibit resistance to mitomycin C can be agentsthat inhibit polypeptides such as the MCRA polypeptide or it can beanalogs of the DNA alkylating or cleaving agents. Preferred examples ofseveral analogs of mitomycins and methods for synthesizing such analogsare disclosed by Kasai et al., cited supra.

Effective amounts of the DNA bioreductive alkylating or cleavage agentor the agent suspected of inhibiting resistance to the DNA alkylating orcleaving agent are those amounts of the agent that would typicallyinhibit the growth of a sensitive cell. Effective amounts of mitomycinare known to those of skill in the art and preferably are 1 to 1,000μg/ml and more preferably about 25 to 100 μg/ml. Effective amounts of anagent suspected to inhibit resistance are preferably within the samerange and can be determined using standard dose response methodology.

Transformed cells are incubated in the presence of the DNA alkylating orcleaving agent and the agent suspected of inhibiting resistance of thecell to the DNA alkylating or cleaving agent. The incubation perioddepends on the assay used to determine whether there has been a changein the resistance of the cell to the DNA alkylating or cleaving agent.If the change in resistance of the cell is measured by DNA-DNAcrosslinking as described by Masuda et al., cited supra, incubation canbe as short as one hour. If the change in resistance of the cell isdetermined by growth of the transformed cells, the incubation period isabout 1 to 10 days. An inability of the transformed cell to grow duringthe incubation period indicates that resistance of the cell to the DNAbioreductive alkylating or cleaving agent has been inhibited.

Determining whether the suspected agent inhibits resistance of the cellto a DNA alkylating or cleaving agent can be accomplished by severalmethods. As described herein, if the transformed cell's resistance tothe DNA alkylating or cleaving agent is inhibited, its growth isinhibited about 10 to 100-fold. If the transformed cell's resistance isinhibited, DNA-DNA alkylation or crosslinking is increased about 2 to20-fold over that of resistant cells.

In an alternative version, an agent suspected of inhibiting resistanceof a cell to a DNA bioreductive alkylating or cleaving agent can beidentified by determining if the suspected agent inhibits the functionof a polypeptide encoded by a DNA sequence that provides resistance tothe agent. The steps of this method involve providing a substantiallypure MCRA polypeptide and a sample of a DNA containing CpG residues. TheMCRA polypeptide and the sample of DNA are then incubated with an agentsuspected to inhibit the function of the MCRA polypeptide and the DNAbioreductive alkylating and cleaving agent. After incubation, it isdetermined whether the suspected agent can inhibit the function of theMCRA polypeptide.

Substantially pure MCRA polypeptide can be obtained from a transformedcell line as described herein. A substantially pure polypeptide is apolypeptide that does not contain other polypeptides as determined bySDS-PAGE. See FIG. 10. A sample of DNA containing CpG residues can beobtained commercially or synthesized by automated DNA synthesis orisolated from a microorganism such as S. lividans. The agent suspectedof inhibiting the MCRA polypeptide can bind to and block the function ofMCRA. Such an agent could be an antibody to MCRA, an analog orderivative of mitomycin or a polypeptide designed to inhibit the bindingor function of the FAD cofactor, e.g., a compound that covalently bindsto or otherwise alters the His⁶⁴ residue of MCRA.

The function of the MCRA is predicted to include deactivation of the DNAalkylating or cleaving agent so that the DNA alkylating or cleavingagent can no longer bind to and/or cleave or alkylate DNA. An inhibitionof the function of the MCRA polypeptide can be determined by whether theDNA bioreductive or alkylating or cleaving agent can bind to DNA and/orcatalyze DNA crosslinks or breaks. Binding of the DNA alkylating orcleaving agent to the DNA and crosslinking or cleaving of DNA can bedetermined by standard methods. If the suspected agent inhibits the MCRApolypeptide, binding of the DNA alkylating or cleaving agent to DNA willincrease about 2 to 10 fold or the DNA-DNA crosslink formation willincrease about 2 to 20 fold.

G. Methods for Identifying DNA Sequences in Other Organisms that areHomologous to the mcr and mrd Gene Loci

The invention also provides a method for identifying DNA sequences ofother organisms that are homologous to the mcr and mrd gene loci thatprovide resistance to mitomycins. Homologous sequences in otherorganisms or cells, especially tumor cells, can be involved in thedevelopment of tumor drug resistance to DNA bioreductive alkylating orcleaving agents such as mitomycins. A preferred method involvesgenerating a DNA library from cells of the organism using standardmethods, followed by amplifying DNA sequences of the library usingpolymerase chain reaction. The polymerase chain reaction (PCR) includesthe use of oligonucleotide primers that are complementary to portions ofthe mcr or mrd DNA sequences. The amplified products are isolated andanalyzed for homology to mcr and mrd DNA sequence by hybridization to aDNA probe complementary to all or a portion of the mcr or mrd DNAsequence.

A DNA library from the cells of the organism can be generated usingstandard methods. The organism is preferably mammalian and the cells arepreferably mitomycin C or multidrug-resistant tumor cells.

The DNA sequences in the DNA library are amplified by standard PCRtechniques as described by Sambrook et al., cited supra. Oligonucleotideprimers are preferably about 20 to 30 nucleotides in length. Thesequence of the primers is selected to be complementary to the DNAcoding sequence of the mcr gene locus or the mrd gene locus. Preferably,two different primers can be used in a single PCR reaction. Thepreferred primers are complementary to DNA sequences of the mcrA genethat encode amino acids 171 to 177:

CCT GTT CTG GGC GGT CCG CGG (SEQ ID NO:8) L   F   W   A   V   R   G (SEQID NO:14)

and the DNA sequence of the mcrA gene that encodes the amino acids 429to 436.

TAC GAC CCG GAC AAC ATG TTC CGA (SEQ ID NO:9)Y   D   P   D   N   M   F   R

Other primers can be selected from other sequences by identifyingregions of the DNA sequence of the mcr or mrd locus that encode aminoacids that are shared by several related proteins such as6-hydroxy-D-nicotine oxidase. Amino acids 171 to 177 and 429 to 436 ofthe MCRA polypeptide are very similar to the amino acids found in6-hydroxy-D-nicotine oxidase. Primers can be prepared by standardmethods such as automated DNA synthesis.

Once primers are selected, they can be synthesized and combined with DNAsequences of the DNA library of the organism in a polymerase chainreaction. Conditions and amounts of material required for the polymerasechain reaction are known to those of skill in art and are described inSambrook et al., cited supra. The polymerase chain reaction results inamplified products complementary to DNA sequences found in the sourceorganism's genome.

The amplified products are then isolated and it is determined whetherthe amplified products are substantially homologous to the mcr or mrdgene loci. Homologous amplified products can be identified byhybridization under low stringency conditions to a DNA probe by standardmethods. Hybridization conditions and DNA probes have been describedherein. An amplified product is substantially homologous if ithybridizes to a DNA probe complementary to a portion of the mcr or mrdgene locus under low stringency conditions and preferably shares about75-100% DNA sequence identity and more preferably, shares about 90-100%DNA sequence identity.

The preferred probes are those that are complementary to all or aportion of the 2.2 kb BlII-SphI DNA sequence from pDHS3005 encoding themcrA and mcrB genes and the 4.2 kb BclI fragment of the pDHS30001encoding the mrd locus.

Hybridization can be detected under both low and high stringencyconditions using standard methods. Identification of such homologous DNAsequences from other organisms or cells can be used to identify agentsthat can inhibit resistance to DNA bioreductive alkylating or cleavingagents as described herein. Sequences that hybridize to the probes canbe analyzed by standard methods such as restriction endonuclease mappingand DNA sequencing.

Polypeptides that are homologous or related to MCRA can also be detectedin other types of resistant cells using antibodies specific for MCRA.For example, antibodies specific for MCRA can be used to identifyhomologous or related polypeptides in mitomycin resistant tumor celllines using ELISA assays. The antibodies can also be used to isolate andpurify related polypeptides using affinity chromatography.

H. Methods of Screening for Novel DNA Bioreductive Alkylating orCleaving Agents

The invention also provides methods for screening for novel DNAbioreductive alkylating or cleaving agents. A method involves incubatingcells that are resistant to a DNA bioreductive alkylating or cleavingagents and detecting induction of expression of a DNA sequence thatimparts resistance to a DNA bioreductive alkylating or cleaving agentsuch as mitomycin C. The cells can be naturally occurring resistantcells such a S. lavendulae or the cells can be transformed with anexpression cassette including a DNA sequence that provides resistance toa cell to a DNA bioreductive alkylating or cleaving agent.

In a perferred version, a cellular extract from a resistant Streptomycesspp is incubated with a transformed cell such S. lividans carryingplasmid pDHS3000 and the cells are grown in the presence of the cellularextract for 48 hours to stationary phase. After 48 hours induction ofmcrA is monitored. Induction of mcrA can be detected by detecting thepresence of mcrA using ELISA as discussed in Example 3. All extractsexhibiting the ability to induce expression of mcrA or mrd can befurther fractionated to identify the compounds that act as inducers ofexpression of resistance. These compounds can include novel DNAbioreductive alkylating or cleaving agents which could be useful asanticancer agents.

EXAMPLE 1 Cloning of Mitomycin C Resistance Genes

Genes encoding resistance to mitomycin C were identified and cloned fromS. lavendulae DNA. Two gene loci were identified and designated mcr andmrd. The gene locus designated mcr encodes mcrA, mrd and ORF3. The genelocus designated mrd was also identified and isolated.

Bacterial Strains and Plasmids

The E. coli strain used was DH5αF′. The S. lividans strain used was 1326(John Innes strain S. lividans 66). Streptoverticillium spp. used inthis study have been identified as Streptomyces lavendulae in accordancewith the proposition that the genus Streptoverticillium be unified withthe members of the genera Streptomyces, in the species lavendulae (Wittet al., System. Appl. Microbiol., 13:361 (1990)). S. lavendulae strainsused in this study were B619, NRRL 2564, KY681, and PB1000. Strain B619was a gift from Abbott Laboratories. Strain PB1000 was derived fromstrain B619 as described below. Strain NRRL 2564 was obtained from theAmerican Type Culture Collection (ATCC 27422). Strain KY681 was kindlyprovided by Kyowa Hakko Kogyo, Co., Ltd.

Strain PB1000 is a highly resistant MMC mutant. Mycelia of S. LavendulaeB619 was plated on MMC gradient plates. Mutants resistant to 250 μg/mlof mitomycin C were obtained. After several rounds of replating,selected mutants were identified with resistance to greater than 1000μg/ml of MMC. One such mutant designated PB1000 was highly resistant toMMC and had a normal morphological phenotype.

The high copy Streptomyces plasmid pIJ702 available from John InnesInstitute, Norwich, England was used for all cloning work performed inStreptomyces lividans. pBR322 and pUC119 were used for all cloning workperformed in E. coli and these can be obtained from commerciallyavailable sources.

Plasmids used to clone mitomycin C resistance genes were isolated andconstructed using standard methods as follows. pDHS3000 and pDHS3001were isolated from MMC resistant colonies obtained as described on pages33-34. Plasmid preparations of MMC resistant colonies revealed twodistinct DNA fragments. Some clones possessed pDHS3000 which contained a6.7 kb insert (mcr) and conferred high level resistance (>100 μg/ml).Clones possessing pDHS3001 contained a 4.2 kb insert (mrd) and conferredlower levels of MMC resistance (25 μg/ml) in liquid culture. See TableI.

pDHS3002 was constructed by the digestion of pDHS3000 with PvuII andClaI, Klenow treatment of the total digestion products, and ligationinto the blunt-ended BglII site of pIJ702. pDHS3003 was constructed bythe digestion of pDHS3000 with BamHI and ClaI, Klenow treatment of thetotal digestion products, and ligation into the blunt-ended BglII siteof pIJ702. pDHS3004 was constructed by the digestion of pDHS3003 withBglII and PstI, Klenow treatment of the total digestion products, gelpurification of the 3.5 kb fragment, and ligation of the purifiedfragment into the blunt-ended BglII site of pIJ702. pDHS3005 wasconstructed by the digestion of pDHS3003 with BglII and SphI, Klenowtreatment of the total digestion products, gel purification of the 2.2kb fragment, and ligation of the purified fragment into the blunt-endedBglII site of pIJ702. pDHS3006 was constructed by the digestion ofpDHS3003 with FspI and StuI, Klenow treatment of the total digestionproducts, gel purification of the 1.9 kb fragment, and ligation of thepurified fragment into the blunt-ended BglII site of pIJ702. pDHS3007was constructed by the digestion of pDHS3003 with NcoI and SphI, Klenowtreatment of the total digestion products, gel purification of the 0.8kb fragment, and ligation of the purified fragment into the blunt-endedBglII site of pIJ702. pDHS3008 was constructed by the digestion ofpDHS3003 with PstI and StuI, Klenow treatment of the total digestionproducts, gel purification of the 1.8 kb fragment, and ligation of thepurified fragment into the blunt-ended BglII site of pIJ702. pDHS3009was constructed by the digestion of pDHS3003 with PstI and NcoI, Klenowtreatment of the total digestion products, gel purification of the 2.2kb fragment, and ligation of the purified fragment into the blunt-endedBglII site of pIJ702. pDHS3010 was constructed by the digestion ofpDHS3000 with BclI, Klenow treatment of the total digestion products,and ligation into the BamHI site pBR322. See Table I.

TABLE I Bacterial strains and plasmids Strains or Plasmids RelevantCharacteristics Source or Reference STRAINS Streptomyces lividans 1326mmc−, mc^(s) Hopwood et al. cited supra. Streptomyces lavendulae B619mmc+, mc^(r) Abbott Laboratories NRRL 2564 mmc+, mc^(r) ATCC KY681 mmc+,mc^(r) Kyowa Hakko Kogyo Inc. PB1000 mmc+,mc^(r) This work Escherichiacoli BRL DH5aF′ PLASMIDS pIJ702 tsr, hyg, mel Hopwood et al., citedsupra. pDHS3000 pIJ702 with 6.7 kb BclI This work DNA insert fromStreptomyces lavendulae B619; contains mcr locus pDHS3001 pIJ702 with4.2 kb BclI This work DNA insert from Streptomyces lavendulae B619;contains mrd locus pDHS3002 pIJ702 with 5.0 kb This work PvuII-ClaIsubclone from pDHS3000 pDHS3003 pIJ702 with 3.0 kb This work BamHI-ClaIsubclone from pDHS3000; contains mcrA, mcrB, and orf 3 pDHS3004 pIJ702with 3.5 kb This work BglII-PstI subclone from pDHS3003; contains mcrA,mcrB, and orf 3 pDHS3005 pIJ702 with 2.2 kb This work BglII-SphIsubclone from pDHS3003; contains mcrA and mcrB pDHS3006 pIJ702 with 1.9kb This work FspI-StuI subclone from pDHS3003; contains mcrA pDHS3007pIJ702 with 0.8 kb This work NcoI-SphI subclone from pDHS3003; containsmcrB pDHS3008 pIJ702 with 1.8 kb PstI- This work StuI subclone frompDHS3003; contains orf 3 pDHS3009 pIJ702 with 2.2 kb PstI- This workNcoI subclone from pDHS3003; contains mcrB and orf 3 pDHS3010 pBR322with 8.3 kb BclI This work subclone from pDHS3000 pBR322 lacZ, bla, tetBRL

Media and Growth Conditions.

Streptomyces spp. were grown in tryptic soy broth (TSB) for DNAisolation, or in yeast extract malt extract (YEME) broth supplementedwith 5 mM MgCl₂ and 5 mM glycine for transformation. Streptomyces weregrown on R2YE agar medium and E. coli was grown on LB agar medium.Growth media were supplemented with the appropriate antibiotics at thefollowing concentrations: Agar plate; ampicillin, 50 μg/ml; mitomycin C,20 μg/ml; thiostrepton, 20 μg/ml: Luria Broth; ampicillin, 50 μg/ml;mitomycin C, 5 μg/ml; thiostrepton, 5 μg/ml.

Bacterial Transformations.

Preparation of S. lividans 1326 protoplasts and their subsequenttransformations were performed as described in Hopwood et al., GeneticManipulations of Streptomyces. A Laboratory Manual, John InnesFoundation, Norwich U.K. (1985). Competent E. coli DH5αF′ were preparedand transformed according to Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1985).

DNA Manipulations and Sequencing.

Isolation and purification of DNA was performed according to Sambrook etal., cited supra. Restriction enzymes were obtained from Gibco/BRL(Gaithersburg, Md.), and digestions were performed according to themanufacturer's instructions. Genomic DNA was isolated from Streptomyceslavendulae B619 using the Kirby protocol described in Hopwood et al.,cited supra. ³²P-labeled DNA probes were generated by nick-translationas described by Sambrook et al., cited supra., and Southern blothybridizations were performed as described by Hopwood et al., citedsupra., using low stringency conditions. For example, afterhybridization in 50% formamide, 5×SSC, 25 mM sodium phosphate, 0.1%sodium pyrophosphate, and 1×Denhardt's solution, washing at 55° willremove any hybridized probe with less than about 76% penology to thebound sequences.

To determine the nucleotide sequence of mcr locus, the 3.2 kb BamHI/ClaIfragment from pDHS3003 was cloned into pUC119. The internal 2.5 kb ApaIfragment was purified using the GeneClean II kit (BIO 101, La Jolla,Calif.) according to the manufacturer's instructions. Differentallotments of this fragment were digested with SalI, BstEII, and RsaI.In addition, the fragment was double digested with XhoI and NcoI. Thedigestion products were blunt-ended using Klenow as described bySambrook et al., cited supra. These products were cloned into the SmaIsite of pUC119. Single-stranded DNA was isolated from clones containinginserts using the helper phage MK037. Single-stranded DNA sequencing wasperformed using the dideoxy chain termination method. (Sequenase kit,version 2.0; deazaguanosine dinucleotides, United States Biochemical;and [α-³⁵S] dATP.) DNA sequence analysis was performed using theIntelligenetics, and the Wisconsin Genetics Computer Group softwareprograms version 7.0 (Madison, Wis.). “FRAME” analysis was performedusing the Intelligenetics Geneworks codon composition DNA algorithm withthe nucleotide range set at 25 base pairs 5′ and 3′ of the DNA sequenceanalyzed. The sequence is shown in FIG. 2.

To determine the sequence for mrd, a 5.5 kb BclI fragment from plasmidpDHS3001 was isolated and subjected to shotgun cloning into pUC119.Briefly, the BclI generated fragments were ligated using T4 DNA ligase.The ligated product was disrupted using sonication to generate randomlysized fragments. Selected fragments ranging in size from 500 to 1000 bpwere isolated via agarose gel electrophoresis. Fragments were clonedinto SmaI site in pUC119. Single stranded DNA was produced and sequencedvia the dideoxy chain termination method of Sanger et al. Analysis ofsequencing gel is being conducted with the aid of computer software“GeneWorks®” by Intelligenetics, Mountain View, Calif., and Frame byGeneWorks from Intelligenetics. The sequence for the mrd (formerly mcrB)locus of S. lavendulae is shown in FIG. 11. The sequence has twocomplete and two partial open reading frames (ORF) as follows: from the5′ end an incomplete reading frame at 1-320 nucleotides; an open readingfrom 1055-1519 nucleotides; an open reading frame from 1871-3376; and anincomplete reading frame from 3376-4052. The current map of the mrdlocus is shown in FIG. 12. The translation product of the ORF at the 3′end of the clone aligns with mycinamicin IV hydroxylase fromMicromonospora griseorubida. A polypeptide having the predicted aminoacid sequence encoded by this ORF shows a strong similarity tomycinamicin hydroxylase.

Identification, Cloning and Sequencing of Mitomycin C Resistance Genes

In order to identify and characterize S. lavendulae DNA that confersresistance to MMC, a S. lavendulae DNA library was constructed byisolating total chromosomal DNA, digesting completely with BclI, andligating into the BglII site in the high copy Streptomyces vector pIJ702as described by Hopwood et al., cited supra. Protoplasts of S. lividans(S. lividans determined to be suitable hosts because of their highsensitivity to MMC in liquid culture, and on agar medium (totalinhibition of growth was observed at 10 μg/ml)) were generated andtransformed using standard methodology.

The sensitivity of S. lividans 1326 was tested for its resistance to MMCby plating spores onto plates containing 1, 10, and 25 μg/ml MMC. Somegrowth was observed at 10 μg/ml MMC, however, growth was not observed at25 μg/ml MMC. B619 genomic DNA was digested with BclI to completion andligated into the BglII site pIJ702. S. lividans protoplasts weretransformed with the ligation mixture and plated onto R2YE agar. After24 hours, regenerated protoplasts were overlaid with 25 μg/mlthiostrepton. Approximately 8000 mitomycin C resistant colonies appearedseveral days later. After the colonies began to sporulate they werereplica-plated onto R2YE containing 25 μg/ml thiostrepton and MMC.

Screening for MMC resistant colonies was accomplished by overlaying theregenerated S. lividans protoplasts with thiostrepton to select forcolonies containing recombinant pIJ702 plasmids. Following several daysof growth and sporulation, colonies were replica-plated onto mediumcontaining 25 μg/ml MMC. Two types of drug resistant colonies wereobtained; the first appeared after 24-48 hours and grew at a normalrate, whereas the second type appeared at the same time but grew at asignificantly slower rate.

Plasmid preparations of MMC resistant colonies revealed two distinct DNAfragments. The clones that grew at a normal rate possessed pDHS3000,which contained a 6.7 kb fragment (mcr) and conferred high levels ofresistance (>100 μg/ml) in liquid medium. The clones that grew at areduced rate possessed pDHS3001, which contained a 4.2 kb DNA insert(mrd) and conferred lower levels of MMC resistance (25 μg/ml) in liquidculture (Table I).

Both plasmids were purified and used individually to re-transform S.lividans, confirming the MMC resistant phenotype. Transformation ofpIJ702 as a control resulted in MMC sensitive S. lividans, establishingthat in each case, cloned DNA alone was responsible for conferring MMCresistance. These results established that S. lavendulae possesses atleast two genetic loci that confer differential levels of MMC resistanceto S. lividans.

DNA sequencing of mcr (FIG. 2) was performed on a 3.2 kb subclone(pDHS3003) of pDHS3000 shown to confer high levels of MMC resistance inS. lividans. Analysis of the sequence using the Geneworks codoncomposition algorithm revealed three open reading frames (ORFsdesignated mcrA, mcrB, and ORF3). The predicted direction oftranscription for mcrA and mcrB is left to right, while ORF3 would betranscribed divergently. The predicted start site for mcrA is an ATGcodon at nucleotide position 131. The predicted stop codon for mcrA islocated at nucleotide position 1475 and a non-coding region of 49 bpseparates it from mcrB. The putative translational start site for mcrBis a GTG codon at nucleotide position 1527, preceded by aribosome-binding site centered ˜8 nucleotides upstream. A characteristicstem loop structure suggests the presence of a rho-dependent terminatorat the 3′-end of mcrB. The 3′ end of ORF3 is separated by a 112 bpnon-coding region from the 3′ end of mcrB. The predicted start site ofORF3 is an ATG codon at nucleotide position 2862.

For sequencing of the mrd locus a 4.2 kb BclI fragment from plasmidpDHS3001 was isolated and subjected to shotgun cloning into pUC119 forsingle stranded DNA sequencing via the dideoxy chain termination methodof Sanger et al. as described at pages 12-13. Analysis of sequencinggels made with the aid of “Geneworks®” by Intelligenetics, Mountainview,Calif. See FIGS. 11 and 12.

Sequence Similarity of mcrA to the Hydroxy-D-nicotine Oxidase fromArthrobacter Oxidans.

Computer-assisted comparison of the deduced product of mcrA with otherproteins revealed significant similarity with the amino-terminal half of6-hydroxy-D-nicotine oxidase (6HDNO) from Arthrobacter oxidans. See FIG.3. Brandsch et al., Eur. J. Biochem., 167:315 (1987). The 6HDNO operatesin the catabolism of nicotine by catalyzing a two electron oxidation ofthe pyridine ring system, and the stereospecific hydroxylation of thecarbon atom adjacent to nitrogen. Significantly, alignment with thededuced mcrA protein sequence includes a co-linear arrangement with theknown FAD-binding site centered at His⁷¹ of the 6HDNO polypeptide chain(Brandsch et al., cited supra.). The His⁷¹ residue of 6HDNO has beenshown to bind FAD covalently, as opposed to the 6-hydroxy-L-nicotineoxidase, which involves a noncovalent FAD-binding site (Mohler et al.,Eur. J. Biochem., 32:1364 (1972). Another significant comparison wasfound with the L-gulonolactone oxidase (L-GLO) from liver microsomes,which involves FAD-mediated oxidation as well (FIG. 4). Interestingly,there is considerable sequence divergence at the carboxy-terminal halfof the mcrA, and 6HDNO, and L-GLO protein sequences.

EXAMPLE 2 Genomic and Transcriptional Analysis of the mcr lociConferring Resistance to MMC

Hybridization of mcr and mrd to Genomic DNA of Independent Isolates ofMMC-producing S. lavendulae.

In order to establish that mcr and mrd were present in MMC-producing S.lavendulae, we performed Southern blot analyses of genomic DNA from fourMMC producing S. lavendulae strains (B619, PB1000, NRRL 2564, KY681), S.lividans 1326 (negative control), using 6.7 kb BclI fragment containingmcr from pDHS3000 and using 4.2 kb BclI fragment containing mrd frompDHS3001 as hybridization probes. See FIG. 5. Distinct DNA bands wereobserved in each of the MMC-producing S. lavendulae strains. Incontrast, S. lividans showed no hybridization signals with either probe.Interestingly, the copy number of mcr showed variability in theMMC-producing strains of S. lavendulae, and corresponded to the level ofMMC resistance exhibited by the specific MMC-producing strains. This isparticularly evident in S. lavendulae strain PB1000, which expressed thehighest level of resistance to MMC and has the highest copy number ofmcr. PB1000 was generated specifically as a highly resistant variant,which was isolated after a strain development protocol using high levelexposure to MMC.

When mrd was used as a probe in the Southern blot described above,hybridizing DNA was observed in each of the MMC-producing S. lavendulaestrains. However, in contrast to mcr, the copy number of mrd appearedinvariant among the MMC producing strains of S. lavendulae. Notably,there is low but significant hybridization between mcr and mrd, which iseasily observed in the lanes containing pDHS3001 and pDHS3000 (FIG. 5),and in the lane containing PB1000 probed with mrd (FIG. 5B).

With the mcr sequence available, it was possible to determine which ofthe three genes were involved in conferring resistance to MMC in S.lividans. A series of subclones were constructed in pIJ702, and theresults showed that mcrA and mcrB together were required (FIG. 6). Theminimum amount of DNA sequence derived from mcrA and mcrB determined toconfer resistance is defined by about a 2.2 kb BglII/SphI fragmentgenerated from pDHS3005 plasmid. Levels of resistance in the constructcontaining these two genes were identical to that shown for pDHS3000 andpDHS3003 (>100 μg/ml).

A series of subclones from mrd locis have been prepared. Briefly, the4.2 kb BclI fragment from pDHS3001 has been subcloned into p1J702(plasmid) as shown in FIG. 12. The subclones have been introduced intoS. lividans and resistance to MMC evaluated as described previously. A3280 bp AflIII/AscI fragment. did confer resistance as well as a 3152 bpNot I fragment. A 1696 bp PvuI/AscI fragment and 1032 bp Not I/PvuIfragment are being evaluated. Resistance was conferred at 25 μg/ml.

RNA Isolation and Transcriptional Start Point Determination.

In order to determine the transcriptional start point for the putativemcrA-mcrB operon, a primer extension experiment was performed. RNA wasextracted from S. lividans 1326 containing pDHS3000 grown in YEME mediumwith the addition of glycine to 5 mM and mitomycin C or thiostrepton toa concentration of 5 μg/ml. The cultures were allowed to grow for 72hours. Additional drug was added to the mitomycin C induced culture at 5μg/ml 35 minutes before RNA extraction. RNA was isolated using the Kirbyprotocol (from Hopwood et al., cited supra.). Primer extension wasperformed using the primer extension system of Promega Biotech(Madison,. Wis.) according to the instructions of the manufacturer. Theoligonucleotide used for the extension reaction was5′-CCACCTCCTGCTCGTCGGCC-3′ (SEQ ID NO: 10), synthesized by KeystoneLaboratories, Inc. (Menlo Park, Calif.).

RNA was isolated and the Northern blot was performed according toHopwood et. al., cited supra. The RNA gel and electrophoresis wasperformed according to the formaldehyde method described by CurrentProtocols (Ausubel et. al., (1989)). The DNA probe used for thehybridization was a BglII/StuI fragment of pDHS3003. It was gel purifiedusing the BIO 101 GeneClean II kit and radiolabeled using the Gibco/BRLrandom primers labeling system (Gaithersburg, Md.).

The primer extension protocol resulted in two primer extension productsdesignated P1 and P2. The primer extension products were sequenced usingdideoxy chain termination method. The results established the presenceof two transcriptional start points, P1 and P2 with an expression ratioof ˜5:1. The transcriptional start point for P1 is also the firstnucleotide (bp 131) of the translational start codon. Thus, the mcrAtranscript represents a leaderless mRNA, a phenomenon that has beendescribed for several other Streptomyces resistance genes. The second,weaker primer extension product (P2) was observed that represents anmRNA with a transcriptional start point at nucleotide 170. Thistranscriptional start point may ensure basal levels of mcrA and mcrBmRNA. Hybridization of mcr to a 1.8 kb band in a Northern blot from S.lavendulae total RNA provides compelling evidence for a polycistronicmRNA including mcrA and mcrB.

EXAMPLE 3 Studies on Inducible Expression of mcrA

Gene expression of mcrA is induced by low levels of MMC. Completion ofthe DNA sequence for mcr suggested strongly that the mcrA gene productrepresented the protein induced after addition of the drug. It wasdetermined whether other compounds in the mitomycin class were capableof inducing expression of the mcrA-mcrB operon.

A series of mitomycins and related compounds shown below were kindlyprovided by Dr. Hiromitsu Saito of Kyowa Hakko Kogyo, Co., Ltd.

Mitomycin X Y Z Mitomycin A OMe OMe H Mitomycin B OMe OH Me Mitomycin CNH₂ OMe H Mitomycin D NH₂ H Me Mitomycin F OMe OMe Me Mitomycin H OMe HMe Porfiromycin NH₂ OMe Me

Individual drugs were added to fermentation flasks of S. lividanscontaining pDHS3000 (see Table 1) at t=0, and growth of the culture wasallowed to proceed for 120 hours. The mycelium was harvested, and a cellextract prepared by sonication. Expression of mcrA was determined bydetecting the 56,000 dalton polypeptide by SDS-PAGE method. The resultsshowed that only mitomycin A (MMA) and MMC were capable of inducingexpression of mcrA at 1 μg/ml. The other mitomycins induced expressionof mcrA at concentrations of 5 μg/ml or greater.

Induction and expression of MCRA was evaluated for dose response tomitomycin C and the other mitomycins as well by detecting the presenceof MCRA using antibodies to MCRA and an ELISA assay. The ELISA assay wasconducted as follows. About 100-200 ul of 1.0-0.5 ug/ml antigen (MCRA)was added to Corning 96 well ELISA plates (cat no. 25801) and incubatedat 37° C. for 2 hrs or 4° C. overnight. The contents were decanted andthe plates were shaken dry. The wells were washed with 1× with PBS Tween20 (0.05% T20). To the wells, 100-200 ul of 0.5% nonfat dry milk in PBSwas added as a blocking agent. The plates were incubated for 1 hour at37° C., the contents were decanted and the plates were shaken dry. Thewells were washed four times with PBS-T20 and were shaken dry. Onehundred microliters of primary antibody, at a dilution of 1:4000 in PBSwith 0.5% nonfat dry milk, was added to the wells. The plates wereincubated at 37° C. for 1 hour. The wells were washed four times withPBS-T20 and were shaken dry. One hundred microliters of secondaryantibody (goat anti-rabbit HRP), at a dilution of 1:3000 in PBS-T20, wasadded to the wells and incubated at 37° C. for 1 hr or RT for 2 hrs. Thecontents were decanted and the plates were shaken dry. The wells werewashed four times with PBS-T20 and were shaken dry. One hundredmicroliters of OPD solution was added to each well. The plates wereagitated for 15 minutes at room temperature.

The results for induction with different amounts of mitomycin C areshown in FIG. 13. The expression of MCRA was MMC concentration dependentand maximum expression of MCRA occurred at concentrations of MMC greaterthan 10 ug per ml (FIG. 13). Concentrations of MMC above this levelresult in a decreased growth rate of the MMC producer S. lavendulae whenit is grown in MMC, which indicates that the mechanism of resistanceconferred by MCRA can be physiologically saturated (data not shown).Significantly, MCRA is induced by concentrations of MMC as low as 0.01ug per ml (30 nM). The induction of MCRA may be induced at a much lowerconcentration physiologically in S. lavendulae.

Since MCRA appeared to provide resistance against other mitomycins, weevaluated those molecules for their ability to induce MCRA expression.Surprisingly, 3 uM of MMA or MMC were the only compounds to induceexpression of MCRA that was visible by SDS-PAGE. However, ELISAsrevealed that MCRA expression was induced by other mitomycins but to alesser degree (FIG. 14). Higher concentrations of the mitomycinsincreased the level of MCRA expression.

This result is significant in view of the structural and biosyntheticrelationship between MMA and MMC. Indeed, MMA is a precursor, andperhaps the earliest isolatable molecule in the pathway. Thus, it isreasonable that MMA would provide a primary induction signal formcrA-mcrB expression in S. lavendulae.

Experiments were performed to evaluate the ability of DNA alkylatingagents to activate MCRA expression in order to determine whetherinduction of MCRA could be due to DNA damage as a result of DNAalkylation. As seen in FIG. 15, neocarzinostatin (NCS), mephalan,(±)-1,2:3,4-diepoxybutane (DEB), and daunomycin did not induce MCRAexpression at 76 mM.

The ability of mcr locus to confer resistance to other mitomycins wasexamined. To determine whether resistance to other mitomycins andmitosanes is conferred by MCRA, we examined the ability of S.lividans/pDHS3000 to grow in the presence of MMC related molecules. Asshown in Table II, resistance against all the compounds tested wasconferred by pDHS3000. However, mitomycin D and H were not lethal to thecontrol culture, S. lividans pIJ702. The non-lethality of mitomycins Dand H does not reflect an inability of mitomycin D and H to penetratecell barriers since these compounds were able to induce MCRA expression.

TABLE II Mitomycin resistance conferred by pDHS3000 Mitomycin S.lividans S. lividans (50 ug/ml) pIJ702 pDHS3000 None + + A − + B − + C− + D + + F − + H + + Porfiromycin − +

EXAMPLE 4 Purification of MCRA

Due to the potential significance of MCRA as a novel resistance protein,the biochemical mechanism of action of the gene product will beinvestigated. The protein was purified to confirm its identity, and toperform a series of in vitro experiments concerning its precisebiological function.

A purification scheme has been developed for MCRA. S. lividanscontaining pDHS3000 was grown to stationary phase in the presence of 5μg/ml MMC. The mycelia was harvested after 54 hours of growth,centrifuged at 5,000 rpm for 10 minutes. The supernatant was decantedand the mycelial pellet resuspended in two volumes of protein extractionbuffer (50 mM Tris.HCl pH 8.0, 10% glycerol, 2 mM EDTA pH 8.0). One halfof the total mycelia was frozen at −80° C. while the other half wasprocessed to isolate MCRA. All of the subsequent steps were performed at4° C. The mycelia were disrupted by passing twice through a French pressat 1500 psi. The homogenate was centrifuged at 10,000 rpm for 1 hour.

The homogenate supernatant was removed and proteins precipitated from itby the gradual addition of ammonium sulfate until 100% saturation. Thesaturated ammonium sulfate solution was allowed to stir overnight andthen centrifuged at 10,000 rpm for 30 minutes. The supernatant wasdiscarded and the pellet was resuspended in protein extraction bufferprecooled to 4° C. The solution was dialyzed against 4 liters of proteinextraction buffer five times over two days. The dialysis tubing wasplaced in a tray containing 200 g of PEG 6000 for 6 hours. Dialysis wasperformed against 4 liters of protein extraction buffer for 4 hours. Theprotein solution was removed from the dialysis tubing and centrifuged at5,000 rpm for 10 minutes. The supernatant was removed and ammoniumsulfate added to a concentration of 50%. The solution was allowed tostir for one hour and then centrifuged at 7,000 rpm for 10 minutes. Thesupernatant was removed and ammonium sulfate was added to bring theconcentration up to 70%. The solution was allowed to stir for one hourand then centrifuged at 7,000 rpm for 10 minutes. The supernatant wasremoved and the pellet was resuspended in protein extraction bufferfollowed by dialysis against 4 L of buffer twice for 12 hours. Thedialysis tubing was placed in a tray containing 100 g of PEG 6000 for 6hours. The dialysis tubing was washed with distilled water and dialyzedovernight against 2 L of 50 mM phosphate buffer, pH 7.0. The dialysatewas centrifuged at 5,000 rpm for 5 minutes. The supernatant was removedand sterile filtered.

This protein solution was loaded onto a DEAE column equilibrated with 50mM phosphate buffer pH 7.0 using an Econosystem (BIORAD). A distinctyellow band could be seen at the head of the column. The column waswashed with 1 L of the same buffer. The column was eluted with 1 L of 0to 0.3 mM KCl gradient; 4 ml fractions were collected. Fractions wererun on SDS-PAGE evaluated for a protein with the ability to co-migratewith the MC inducible protein from S. lividans 1326/pDHS3000 MMC inducedcell extracts. Fractions containing the MCRA protein were concentratedusing Centriprep (Amicon, Beverly Mass.) with a MW cut off of 30,000according to instructions from the manufacturer. The concentratedprotein solution was a bright yellow color. Gel filtration (SephadexHR200, Pharmacia) was performed to further purify MCRA. The column(size) was washed with 50 mM phosphate buffer, pH 7.0 and loaded with 2mls of the protein solution. The column was run at a flow rate of 0.2mls/min. and 4 ml fractions collected. Fractions were analyzed bySDS-PAGE and revealed that the protein co-migrating with the MCinducible protein was substantially pure.

In an effort to isolate highly purified MCRA, a BIORAD prep cellemploying SDS-PAGE was used. A 10% resolving gel (Gibco/BRL) was pouredand the protein eluted using running buffer (Gibco/BRL). Fractions werecollected and analyzed for the protein MCRA. Fractions containing pureMCRA were pooled and concentrated to 0.5 milliliter using centriprep 30.The buffer of the solution was changed by diluting the protein solutionin PBS and concentrating. This was repeated twice. This protein solutionwas used for the production of polyclonal antibodies, N-terminalsequencing, mass and ultraviolet spectroscopy.

The purified MCRA protein is about a 56,000 dalton protein on SDS-PAGEas shown in FIG. 10.

N-terminal sequence analysis of the MCRA protein was conducted bystandard methods using an Applied Biosystems, Inc. 476 Sequencer (pulseliquid mode). Data analysis was done with the AB1 610 Sequence AnalysisSoftware. The analysis shown below shows that the sequence of theisolated MCRA polypeptide agrees with the predicted amino acid sequencefrom the nucleotide sequence:

Predicted MCRA Sequence: MSTQWGWALEPDQPGY (SEQ ID NO: 12)

N-terminal Sequence Analysis MCRA: MSTQWGWALEPD (SEQ ID NO: 16)

Final purification of MCRA yielded a yellow protein, the UV spectrum ofwhich suggested that the protein was covalently modified by theco-factor FAD (FIG. 16).

Covalent attachment of FAD was verified using several methods. The laststep in the purification strategy for MCRA made use of a preparativeSDS-PAGE cell which consequently denatured MCRA. If the co-factor FADwas non-covalently bound it would dissociate from the protein duringthis procedure. The results show that the molecular weight of thepolypeptide was not altered by denaturation. These results indicate thatFAD is covalently bound to MCRA.

In order to further verify that FAD was covalently bound to MCRA, lineartime of flight and electrospray mass spectroscopy were performed on themodified MCRA protein. For linear time of flight mass spectroscopy, MCRAwas dissolved in a solution of 50 nmol sinapinic acid in a 1:1 waterethanol mix (vol./vol.) with 0.1% Trifluoroacetic acid (TFA) at a finalconcentration of 0.3 pmol. The solution was dried on a sample slide andallowed to form a matrix. The matrix containing MCRA was analyzed by aKratos (Manchester, UK) Komapact Maildi III using high power and 81laser shots. Electrospray mass spectroscopy was performed by Cedric H.L. Shackleton, Ph.D., D.Sc. at the Mass Spectrometry Facility of theChildren's Hospital Medical Center of Northern California.

The time of flight data revealed that the protein solution contained asingle protein with the molecular weight of 49,244-Sinapinil acid(207)=49,037. While the electrospray data revealed a protein withmolecular weight, 49,005 plus the exact mass of FAD. Both of theseresults clearly demonstrate that the MCRA protein with the N-terminalmethionine removed, is covalently modified by FAD.

EXAMPLE 5 Development of Antibodies to MCRA

Once MCRA was isolated and purified, polyclonal antibodies wereprepared. Monoclonal antibodies can be prepared by standard methods asdescribed below.

Anti-MCRA polyclonal antibodies were produced as follows. 500 ug ofpurified MCRA was mixed in a ratio of 1:2 with complete Freunds adjuvantto homogeneity. The mixture formed an emulsion when passed back andforth through two connected syringes. Two New Zealand white rabbits wereeach inoculated with a suspension containing 500 ug of MCRA. After twoweeks they were inoculated again and one month later they werereinoculated. After seven days, blood was removed from the rabbits andthe serum was titered for antibodies against MCRA by ELISA assay. Thepolyclonal antibody titer was 1:8000. The ELISA assay was conducted asdescribed in Example 3.

Monoclonal antibodies can be formed using the standard Kohler, Milsteintechnique. Pursuant to the Kohler, Milstein technique, immunization ofthe mammalian host is accomplished within this dose parameter bysubcutaneous or intraperitoneal injection of the immunogen compound inadjuvant. Administration is repeated periodically and preferably for atleast four injections. Three days before the spleen is removed, apriming injection of immunogen compound is again administered. Aftertheir separation, the spleen cells are fused with immortal mammal cellssuch as mouse myeloma cells using the techniques outlined by Kohler andMilstein. Polyethylene glycol (PEG) or electrical stimulation willinitiate the fusions. The fused cells are then cultured in cell wellsaccording to culture techniques known in the art. Cellular secretions inthe culture medium are tested after an appropriate time for the presenceof the desired cellular products.

The selection technique for identifying the appropriate monoclonalantibody is an important aspect for determining the immunospecificity ofthe monoclonal antibody. The selection techniques call for determiningthe binding affinity of the hybridoma cellular products for themitomycin C resistance polypeptide MCRA and against cross-reactivecontrols. In particular, hybridoma culture fluid is tested in screeningassays for reactivity with mitomycin C resistance polypeptide MCRA andlack of immunoreactivity with bovine serum albumin.

Screening assays can be performed by immunoenzymatic-assay,immunofluorescence, fluorescence-activated cell sorter,radioimmunoassay, immuno-precipitative assay or inhibition of biologicalactivity. The hybridoma cultures selected will exhibit strong bindingcharacteristics to the MCRA polypeptide and exclude binding with avariety of controls, including BSA and mcrB.

Following the identification of cell cultures producing the desiredmonoclonal antibodies, subcloning to refine the selected culture can beperformed. These techniques are known to those skilled in the art. See,for example, Goding, James Goding, Monoclonal Antibodies: Principles andPractice, 2 nd Edition, Academic Press, San Diego, Calif. (1986), thedisclosure of which is incorporated herein by reference.

Briefly, the appropriately selected cell culture is separated intoone-cell units which are then recultured. The subclone cultures are thenagain tested for specific immunoreactivity, lack of cross-reactivity,and the amount of monoclonal antibody secreted. Those subculturesexhibiting the highest amounts of secreted monoclonal antibody arechosen for subsequent pilot development.

The antibodies specific for MCRA can be used in a variety of assaysincluding ELISA assays and Western blot assays. The antibodies areuseful for analyzing induction of MCRA in cells and identifying otherrelated mitomycin resistance proteins and to analyze the functionaldomains of MCRA.

Western blots of cells expressing MCRA were conducted as follows.Completely dry filter with protein. Dip the filter for 2 sec into 100%Methanol and then soak in 1×TBS (Tris buffered saline) (20 mM Tris.HClpH 7.5, 150 mM NaCl) for 2 minutes. Gently shake at room temperature inblocking solution (5% non-fat dry milk in TBS). Wash 3×10 minutes washsolution (0.1% non-fat dry milk in TBS). Add primary antibody toantibody incubation solution (1% non-fat dry milk, 0.05% Tween-20 inTBS) at 1:8000 and incubate for 2 hours at room temperature on orbitalshaker. Wash 3×10 minutes wash solution. Add secondary antibodyconjugate in TBS +0.05% Tween-20 (1:2000 goat anti-rabbit HRP Zymed).Incubate 2 hours on gentle shaker. Wash 3×10 minutes wash solution.Incubate with color development solution 25 mM Tris.HCl pH 7.5,4-Chloro-1-napthol (3 mg/ml MeOH) 30% H2O2). Rinse with distilled H₂Oand blot with Whatman paper to dry and store.

EXAMPLE 6 Cloning and Analysis of Cosmids Containing DNA Adjacent tomcrA and mcrB; Generation of Mutants of mcrA Blocked in MMC Resistance

In order to establish whether DNA adjacent to one or both MMC resistancegenes includes a cluster of biosynthetic genes for the metabolite, wehave generated a S. lavendulae genomic library using the Streptomyces-E.coli shuttle vector, pNJ1 (Tuan et. al., 1990). High molecular weightgenomic DNA was subjected to partial digestion to generate fragmentsabout 30-40 kb in length. The library was subsequently screened with³²p-labeled mcr or mrd. A single cosmid clone (encompassing ˜25 kb) wasidentified using mcr as a probe, and its map is shown in FIG. 7. Formrd, a series of overlapping cosmid clones (encompassing ˜40 kb) wereidentified and mapped, as shown in FIG. 8. It will now be possible tochoose specific regions of the cosmid clones surrounding the resistancegene mcr or mrd to begin probing for genes involved in biosynthesis ofMMC.

Analysis of S. lavendulae DNA involved in MMC biosynthesis involves themethod of gene disruption. In order to determine whether mcrORF3 isinvolved in a biosynthetic step for MMC biosynthesis, we decided togenerate a clone to test functional activity by gene disruption.Therefore, mcrORF3 was used to generate a clone in pUC119 useful forgene disruption in S. lavendulae. The thiostrepton resistance gene (tsr)was inserted into the middle of a fragment internal to the predictedmcrORF3 reading frame. Single-stranded DNA generated from this constructwas used to transform S. lavendulae protoplasts. Selection forthiostrepton resistance allowed convenient identification of severalclones that are currently under analysis (FIG. 9). Initially theseclones will be screened for the presence of tsr in the chromosome andintegration of the construct used for gene disruption. Fermentation andanalysis of MMC production will then be performed on clones of S.lavendulae in which the mcrORF3 gene is inactivated.

A similar strategy will be followed for DNA adjacent to mcr and mrdcontained on cosmid clones. Clones with these inserts in pUC119 areidentified and used to transform S. lavendulae. Transformants will bescreened for loss of production of MMC by identifying clones that nolonger exhibit biological activity and then performing HPLC analysis toshow loss of production of MMC.

Another strategy for obtaining S. lavendulae mutants blocked in MMCproduction includes transposon mutagenesis method for Streptomyces spp.This approach involves use of a hypertransposing element Tn5099-10 anda-temperature sensitive delivery system from pGM160. Briefly, the vectorcontaining Tn5099-10 will be transformed into the MMC-producing S.lavendulae wild-type strain. Incubation at elevated temperatures inducestransposition randomly into the chromosome. Transposon mutants blockedin MMC production will be identified using a biological assay asdescribed previously. Loss of MMC production will be confirmed byanalysis of extracts by HPLC.

These methods should provide MMC blocked mutants.

EXAMPLE 7 Method for Identifying Agents that Can Inhibit Resistance of aCell to a DNA Alkylating or Cleaving Agent

A method for identifying agents that inhibit resistance of a cell to aDNA alkylating or cleaving agent can be conducted as described below.

Transformed cultures of S. lividans carrying plasmid pDHS3005 can becultured in the presence of 0, 1, 10, 25, 100, 500, or 1000 μg/ml ofmitomycin C and in the presence or absence of a suspected inhibitoryagent. The transformed cells can be incubated for about 7 days. Afterincubation, growth of the transformed cells is determined by dry cellweight or plate count method. Cells that are resistant to mitomycin Cwill grow in the presence of at least about 10 μg/ml of mitomycin C.Inhibition of resistance of cells to mitomycin is seen when cells cannotgrow in the presence of about 10 to 100 μg/ml of mitomycin C. Asensitive cell will preferably not show any growth at 25 μg/ml ofmitomycin C. It is likely that an inhibitory agent will prevent orinhibit growth of the transformed S. lividans in the presence ofconcentrations of mitomycin of 10 μg/ml or greater, preferably growth isinhibited at least about 10-fold.

EXAMPLE 8 Determination of Mechanism of Action of MCRA

An assay for catalytic activity and to ascertain the mechanism ofresistance toward MMC was developed. A UV spectrophotometric assay hasbeen developed using the absorbance of MMC at 363 nm to determinewhether reduction of MMC is prevented by MCRA. In this assay, freshlyprepared extracts of S. lividans/pDHS3000 containing overexpressed MCRAwere used in a reaction mixture containing MMC and the chemical reducingagent sodium dithionite. Oxidation state of MMC is assessed at 363 nm.The results show clearly that MMC is reduced to the hydroquinone in theabsence of MCRA by a 100% loss of 363 nm (change) in absorbance of MMC.However, when an extract containing overexpressed MCRA is added to thereaction mixture, no change in absorbance (363 nm) of MMC is observed.These experiments indicate that MCRA acts in vivo by protecting S.lavendulae from the adverse affects of MMC through maintaining thenon-activated oxidative state of the molecule.

EXAMPLE 9 Determining Expression of MCRA in a S. lavendulae, a NaturallyResistant Host Cell

The expression of MCRA in S. lavendulae was detected using an ELISAassay on cell lysates taken at various time points during growth of thecells in culture.

Large scale fermentation of S. lavendulae. S. lavendulae was inoculatedinto production media IM-1 as previously described with the exceptionthat the glycerol concentration was doubled. Bioreactor pH wascontrolled by the addition of 6M NaOH and 50% phosphoric acid. Foamingwas controlled by the addition of 10% antifoam when necessary.Bioreactor pressure was maintained at 4 atm. and sparged at 45 psi at 4L/min. The impeller speed was initially 350 rpm at inoculation andincreased to 500 rpm after 24 hours. 50 milliliter samples were takenfrom the reactor for dry weight, MC production, and MCRA expressionmeasurements every 12 hours. 30 milliliters of the sample was processedto isolate the fermentation broth and mycelia. Two, 10 milliliteraliquots of the original sample were processed for dry weightdeterminations.

Samples were centrifuged at 5K for 10 minutes. For dry weightdeterminations the pellets were washed 4 times with 5 volumes ofdistilled water to remove salts and calcium carbonate. Mycelia forprotein isolation was washed 4 times with phosphate buffered saline(PBS). Samples for dry weights were resuspended in 1 volume of distilledwater and poured into pre-weighed aluminum cupcake tins which were driedat 80° C. for 24 hrs after which they were re-weighed and the dry massdetermined. Fermentation broth was sterile filtered at room temperatureand the presence of MMC was assayed by HPLC immediately. Samples forprotein isolation were resuspended in one volume of PBS. The myceliawere incubated on ice for 15 minutes and then sonicated as described.The cell extract was centrifuged at 4° C. at 10K for 30 minutes. Thesupernatant was removed and sterile filtered through a 0.2 uM filter.These extracts were frozen at −80° C. The cell extracts were analyzedfor the presence of MCRA.

S. lavendulae has the ability to produce MMC at high concentrations whenit is grown in the proper fermentation medium. Given this ability it isexpected that it would be resistant to MMC at high levels. Additionally,if MCRA confers MMC resistance then it should be detected in cellsproducing MMC. As shown in FIG. 17, S. lavendulae was grown tostationary phase and produced MMC as determined by HPLC analysis. Inaddition, ELISA assays revealed that MCRA was expressed. MCRA wasexpressed at a low level prior to MMC production, and was stronglyexpressed just prior to MMC biosynthesis. Most likely the induction isdue to the presence of MMC produced which is below the limit of ourability to detect MMC. Thus, MCRA is produced in the naturally resistanthost cell that produces MMC and is inducibly expressed by MMC as hasbeen observed in S. Lividans pDHS3000.

EXAMPLE 10 Assay to Identify Polypeptides Related to MCRA Present in MMCResistant Tumor Cells

Polyclonal and monoclonal antibodies can be used to identify and/orisolate polypeptides related to MCRA present in MMC resistant tumorcells.

MMC resistant tumor cell lines have been described by Pan et al. Dr.SuShu PAN, University of Maryland Cancer Center, The New Frank BresslerResearch Laboratories, Baltimore, Md. 21201 and are available from herlaboratory. Tumor cells can be exposed to varying concentrations of MMCand then cell extracts containing cytoplasmic proteins can be obtained.The cell extracts can be screened for polypeptides reactive withpolyclonal and/or monoclonal antibodies specific for MCRA in an ELISA asdescribed previously for S. lavendulae cell extracts in Example 3. Celllines exhibiting positive reactivity with antibodies to MCRA can befurther analyzed using affinity chromatography and/or Western blotsusing standard methods. It is expected that the antibodies to MCRA wouldidentify mammalian polypeptides related to MCRA. These antibodies can beused to isolate these polypeptides with affinity chromatography.

These antibodies could be useful in isolation and identification of DNAor cDNA sequence coding for a polypeptide related to MCRA using methodsknown to those of skill in the art, such as dot blot, mammalian cDNAlibrary generation, and sequence analysis.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

While the present invention has been described in connection with thepreferred embodiment thereof, it will be understood many modificationswill be readily apparent to those skilled in the art, and thisapplication is intended to cover any adaptations or variations thereof.It is manifestly intended this invention be limited only by the claimsand equivalents thereof.

18 1 1474 DNA Streptomyces lavendulae CDS (131)...(1474) 1 gatcttcctcgttttgggga ggtgctgacg agccggcctt cgcgccgggc ttccccgcgg 60 gcaggcgctcccacaaccat gtccgattcc ttcaagatgc cgccttgagc tgcttgatta 120 catggcgcgcatg agc acg caa tgg gga tgg gcc ctt gag ccg gac cag 169 Met Ser Thr GlnTrp Gly Trp Ala Leu Glu Pro Asp Gln 1 5 10 ccg gga tac gac gac gcc cggctg gga ctg aac cgg gcg gcc gaa tcg 217 Pro Gly Tyr Asp Asp Ala Arg LeuGly Leu Asn Arg Ala Ala Glu Ser 15 20 25 cgg ccg gcc tac gtg gtc gag gcggcc gac gag cag gag gtg gcc gcc 265 Arg Pro Ala Tyr Val Val Glu Ala AlaAsp Glu Gln Glu Val Ala Ala 30 35 40 45 gcg gtg agg ctg gcc gcc gag cagaaa cgg ccc gtg ggt gtg atg gcc 313 Ala Val Arg Leu Ala Ala Glu Gln LysArg Pro Val Gly Val Met Ala 50 55 60 acc ggt cac gga ccg tcc gtg tcg gccgac gac gcc gtg ctg gtc aac 361 Thr Gly His Gly Pro Ser Val Ser Ala AspAsp Ala Val Leu Val Asn 65 70 75 acg cgg cgg atg gaa ggt gtg agc gtt gacgcg gcc cgc gcg acg gca 409 Thr Arg Arg Met Glu Gly Val Ser Val Asp AlaAla Arg Ala Thr Ala 80 85 90 tgg atc gaa gcc ggg gca cgc tgg cgg aag gtgctg gaa cac acc gct 457 Trp Ile Glu Ala Gly Ala Arg Trp Arg Lys Val LeuGlu His Thr Ala 95 100 105 ccg cac ggg ctc gcg ccg ctg aac ggc tcg agcccc aac gtg ggc gct 505 Pro His Gly Leu Ala Pro Leu Asn Gly Ser Ser ProAsn Val Gly Ala 110 115 120 125 gtc ggc tat ctg gtc ggc ggc ggc gcg ggactg ctg ggc cgc cgg ttc 553 Val Gly Tyr Leu Val Gly Gly Gly Ala Gly LeuLeu Gly Arg Arg Phe 130 135 140 ggc tac gcc gcc gac cac gta cgg cgg ctgcgc ctg gtc acc gcc gac 601 Gly Tyr Ala Ala Asp His Val Arg Arg Leu ArgLeu Val Thr Ala Asp 145 150 155 ggc cgc ttg cgc gac gtg acg gcc ggg accgac ccc gac ctg ttc tgg 649 Gly Arg Leu Arg Asp Val Thr Ala Gly Thr AspPro Asp Leu Phe Trp 160 165 170 gcg gtc cgc ggc ggc aag gac aac ttc ggcctg gtc gtg ggc atg gag 697 Ala Val Arg Gly Gly Lys Asp Asn Phe Gly LeuVal Val Gly Met Glu 175 180 185 gtc gac ctg ttc ccg gtc acc cgg ctc tacggc gga ggg ctc tac ttc 745 Val Asp Leu Phe Pro Val Thr Arg Leu Tyr GlyGly Gly Leu Tyr Phe 190 195 200 205 gcg ggc gag gcc acc gcc gag gtg ctgcac gcc tac gcc gag tgg gtc 793 Ala Gly Glu Ala Thr Ala Glu Val Leu HisAla Tyr Ala Glu Trp Val 210 215 220 cgg cac gtg ccc gag gag atg gcg tcctcc gtg ctg ctc gtc cac aac 841 Arg His Val Pro Glu Glu Met Ala Ser SerVal Leu Leu Val His Asn 225 230 235 ccc gac ctg ccc gac gtc ccg gaa ccgctg cgc gga cgc ttc atc acc 889 Pro Asp Leu Pro Asp Val Pro Glu Pro LeuArg Gly Arg Phe Ile Thr 240 245 250 cac ctc cgc atc gcc tac agc ggc gaaccg gca gac ggc gag cac ttg 937 His Leu Arg Ile Ala Tyr Ser Gly Glu ProAla Asp Gly Glu His Leu 255 260 265 gtg cgg ccg cta cgc gaa ctc gga cccatc ctc ctc gac acc gtg cgg 985 Val Arg Pro Leu Arg Glu Leu Gly Pro IleLeu Leu Asp Thr Val Arg 270 275 280 285 gac atg ccc tac gcc gag gtc ggcacg att cat cac gag ccc acg tcc 1033 Asp Met Pro Tyr Ala Glu Val Gly ThrIle His His Glu Pro Thr Ser 290 295 300 atg ccg tac gtc gcg tac gac cgcaac gtg ttg ctg agc gac ctg acc 1081 Met Pro Tyr Val Ala Tyr Asp Arg AsnVal Leu Leu Ser Asp Leu Thr 305 310 315 gac gat gcc gtc gac atc atc gtcgcc ctg gcc gga ccg gac gca ggg 1129 Asp Asp Ala Val Asp Ile Ile Val AlaLeu Ala Gly Pro Asp Ala Gly 320 325 330 gcg ccg ttc gtc acc gaa ctg cggcac ttc ggc ggc gcg tac gcc cgt 1177 Ala Pro Phe Val Thr Glu Leu Arg HisPhe Gly Gly Ala Tyr Ala Arg 335 340 345 ccg ccg aag gtc ccc aac tgc gtgggc ggg cgc gac gcg gcc ttc tcg 1225 Pro Pro Lys Val Pro Asn Cys Val GlyGly Arg Asp Ala Ala Phe Ser 350 355 360 365 ctc ttc acg ggc gcc gtc ccggaa gcc gag ggt ctc cgg cgc cgt gat 1273 Leu Phe Thr Gly Ala Val Pro GluAla Glu Gly Leu Arg Arg Arg Asp 370 375 380 gac ctg ctc gac cgg ctg cgccca tgg agc acc ggc ggc acg aac ctc 1321 Asp Leu Leu Asp Arg Leu Arg ProTrp Ser Thr Gly Gly Thr Asn Leu 385 390 395 aat ttc gcc ggt gtc gag gacatc agc ccg gcg agc gtg gaa gcc gcc 1369 Asn Phe Ala Gly Val Glu Asp IleSer Pro Ala Ser Val Glu Ala Ala 400 405 410 tac act ccg gct gat ttc gcccgg ttg agg gct gtc aag gcc caa tac 1417 Tyr Thr Pro Ala Asp Phe Ala ArgLeu Arg Ala Val Lys Ala Gln Tyr 415 420 425 gac ccg gac aac atg ttc cgagtc aac ttc aac att ccg ccg gcg gag 1465 Asp Pro Asp Asn Met Phe Arg ValAsn Phe Asn Ile Pro Pro Ala Glu 430 435 440 445 tct tgg acg 1474 Ser TrpThr 2 386 DNA Streptomyces lavendulae CDS (53)...(367) 2 tgagcgtagcgaccgagcct gtccggcgtc gtcatggaga gaaggagtcc gg gtg acc 58 Val Thr 1 tcatcc gac gga tcg gac ctc acc act ctg gtc aac gtg ggc cgg tcc 106 Ser SerAsp Gly Ser Asp Leu Thr Thr Leu Val Asn Val Gly Arg Ser 5 10 15 gtg gcgagg tac ttc gag cgc atc ggc atc acc gag atc gcg caa ctg 154 Val Ala ArgTyr Phe Glu Arg Ile Gly Ile Thr Glu Ile Ala Gln Leu 20 25 30 cgg gac cgcgat ccg gtc gag ttg tac gag cgg atg tca gcc gcc ttc 202 Arg Asp Arg AspPro Val Glu Leu Tyr Glu Arg Met Ser Ala Ala Phe 35 40 45 50 ggg cag cgcctc gat ccc tgc ctg ctc gac acc gtc atg tcg gcg gtg 250 Gly Gln Arg LeuAsp Pro Cys Leu Leu Asp Thr Val Met Ser Ala Val 55 60 65 gac cag gcc gaaggc ctg ccc gct cgc ccc tgg tgg cac tac acc ccg 298 Asp Gln Ala Glu GlyLeu Pro Ala Arg Pro Trp Trp His Tyr Thr Pro 70 75 80 gag cgc aag cgg ttgctg gca ggc gaa ggc cat gac cgg gcc ggt gga 346 Glu Arg Lys Arg Leu LeuAla Gly Glu Gly His Asp Arg Ala Gly Gly 85 90 95 acc gcg ggg gag ggg acagcg tagagacaca gccgtgagt 386 Thr Ala Gly Glu Gly Thr Ala 100 105 3 1085DNA Streptomyces lavendulae 3 gaaggggaaa cggcaggaga gggccggcccggcacggagt ggcccgtcag caatggccgg 60 acgggttcac accgccggtg tacgcgagcggccgggtcac ctctccacca gttcgctcag 120 gcttgagggg cggtagaggg catgtagtgcgcggtctact cggaccaggc gcttcgggcg 180 gtcgcagcgg cagaggcgct tcccctgtggccgcgcccgg tccgcgtccc gcgcgacgta 240 cgcgtcctgt gcccggtccc agaacccaggcatcggcttc cgccggtccc ttgccgccac 300 ctcgacggcg cgcggctctg ggtggctcagccgccagcga cactgcctcg ctggccgaag 360 ctccctgtgg tgctgacgag cgtcctcgcccagctgccca aggcgcagcc ggcgctgcgg 420 cgttacggtc taggagctcc cgccgccggcccaggccggc ggcggcagct gcgcaagcca 480 ctctgccgcg cagggccgcg gcccggtctccagccgctct agctgagcga ccagggcgtc 540 gagtacgcgc ccctacggct cgtcccgcccccgctacggc cccctcttcg cctgtgggtg 600 ggtcttcggg gccactcacg acatccgcgaacatagcccg ggcctctgct cctcggccgg 660 acggcaggtg tcgtccagct acgtcggcagtccattgtgc cagcagcaga gcggcggtgg 720 ccatgtgggc ccgcgaggga cgtgggtcggctagcgcggg ccgtcggccc tgcacgcgag 780 aagcatgagc cccctgtgcg acacttaaaatgcggagcgc ctgtctacga gtactccacc 840 gcgggggcca agaccacggc agcacgcggtcccgttcccg actcacggcg tccgccgcac 900 agccttcacg gctacaactg tgtgcgagggcgtgccggcg taggagccta tctcgattac 960 gacaaaacac ctatatctgt gggcaaaatggagtagtagt cgccatacgg cctcggccct 1020 agtccataag gcaggcgtac ggtcactcctcgcagtaccg catggttgcc ctttgcaggt 1080 gccta 1085 4 458 PRT Arthrobacteroxidans 4 Val Ser Ser Lys Leu Ala Thr Pro Leu Ser Ile Gln Gly Glu ValIle 1 5 10 15 Tyr Pro Gln Asp Ser Gly Phe Asp Ala Ile Ala Asn Ile TrpAsp Gly 20 25 30 Arg His Leu Gln Arg Pro Ser Leu Ile Ala Arg Cys Leu SerAla Gly 35 40 45 Asp Val Ala Lys Ser Val Arg Tyr Ala Cys Asp Asn Gly LeuGlu Ile 50 55 60 Ser Val Arg Ser Gly Gly His Asn Pro Asn Gly Tyr Ala ThrAsn Asp 65 70 75 80 Gly Gly Ile Val Leu Asp Leu Arg Leu Met Asn Ser IleHis Ile Asp 85 90 95 Thr Ala Gly Ser Arg Ala Arg Ile Gly Gly Gly Val IleSer Gly Asp 100 105 110 Leu Val Lys Glu Ala Ala Lys Phe Gly Leu Ala AlaVal Thr Gly Met 115 120 125 His Pro Lys Val Gly Phe Cys Gly Leu Ala LeuAsn Gly Gly Val Gly 130 135 140 Phe Leu Thr Pro Lys Tyr Gly Leu Ala SerAsp Asn Ile Leu Gly Ala 145 150 155 160 Thr Leu Val Thr Ala Thr Gly AspVal Ile Tyr Cys Ser Asp Asp Glu 165 170 175 Arg Pro Glu Leu Phe Trp AlaVal Arg Gly Ala Gly Pro Asn Phe Gly 180 185 190 Val Val Thr Glu Val GluVal Gln Leu Tyr Glu Leu Pro Arg Lys Met 195 200 205 Leu Ala Gly Phe IleThr Trp Ala Pro Ser Val Ser Glu Leu Ala Gly 210 215 220 Leu Leu Thr SerLeu Leu Asp Ala Leu Asn Glu Met Ala Asp His Ile 225 230 235 240 Tyr ProSer Val Phe Val Gly Val Asp Glu Asn Arg Ala Pro Ser Val 245 250 255 ThrVal Cys Val Gly His Leu Gly Gly Leu Asp Ile Ala Glu Arg Asp 260 265 270Ile Ala Arg Leu Arg Gly Leu Gly Arg Thr Val Ser Asp Ser Ile Ala 275 280285 Val Arg Ser Tyr Asp Glu Val Val Ala Leu Asn Ala Glu Val Gly Ser 290295 300 Phe Glu Asp Gly Met Ser Asn Leu Trp Ile Asp Arg Glu Ile Ala Met305 310 315 320 Pro Asn Ala Arg Phe Ala Glu Ala Ile Ala Gly Asn Leu AspLys Phe 325 330 335 Val Ser Glu Pro Ala Ser Gly Gly Ser Val Lys Leu GluIle Glu Gly 340 345 350 Met Pro Phe Gly Asn Pro Lys Arg Thr Pro Ala ArgHis Arg Asp Ala 355 360 365 Met Gly Val Leu Ala Leu Ala Glu Trp Ser GlyAla Ala Pro Gly Ser 370 375 380 Glu Lys Tyr Pro Glu Leu Ala Arg Glu LeuAsp Ala Ala Leu Leu Arg 385 390 395 400 Ala Gly Val Thr Thr Ser Gly PheGly Leu Leu Asn Asn Asn Ser Glu 405 410 415 Val Thr Ala Glu Met Val AlaGlu Val Tyr Lys Pro Glu Val Tyr Cys 420 425 430 Arg Leu Ala Ala Tyr LysArg Glu Tyr Asp Pro Glu Asn Arg Phe Arg 435 440 445 His Asn Tyr Asn IleAsp Pro Glu Gly Ser 450 455 5 448 PRT Streptomyces lavendulae 5 Met SerThr Gln Trp Gly Trp Ala Leu Glu Pro Asp Gln Pro Gly Tyr 1 5 10 15 AspAsp Ala Arg Leu Gly Leu Asn Arg Ala Ala Glu Ser Arg Pro Ala 20 25 30 TyrVal Val Glu Ala Ala Asp Glu Gln Glu Val Ala Ala Ala Val Arg 35 40 45 LeuAla Ala Glu Gln Lys Arg Pro Val Gly Val Met Ala Thr Gly His 50 55 60 GlyPro Ser Val Ser Ala Asp Asp Ala Val Leu Val Asn Thr Arg Arg 65 70 75 80Met Glu Gly Val Ser Val Asp Ala Ala Arg Ala Thr Ala Trp Ile Glu 85 90 95Ala Gly Ala Arg Trp Arg Lys Val Leu Glu His Thr Ala Pro His Gly 100 105110 Leu Ala Pro Leu Asn Gly Ser Ser Pro Asn Val Gly Ala Val Gly Tyr 115120 125 Leu Val Gly Gly Gly Ala Gly Leu Leu Gly Arg Arg Phe Gly Tyr Ala130 135 140 Ala Asp His Val Arg Arg Leu Arg Leu Val Thr Ala Asp Gly ArgLeu 145 150 155 160 Arg Asp Val Thr Ala Gly Thr Asp Pro Asp Leu Phe TrpAla Val Arg 165 170 175 Gly Gly Lys Asp Asn Phe Gly Leu Val Val Gly MetGlu Val Asp Leu 180 185 190 Phe Pro Val Thr Arg Leu Tyr Gly Gly Gly LeuTyr Phe Ala Gly Glu 195 200 205 Ala Thr Ala Glu Val Leu His Ala Tyr AlaGlu Trp Val Arg His Val 210 215 220 Pro Glu Glu Met Ala Ser Ser Val LeuLeu Val His Asn Pro Asp Leu 225 230 235 240 Pro Asp Val Pro Glu Pro LeuArg Gly Arg Phe Ile Thr His Leu Arg 245 250 255 Ile Ala Tyr Ser Gly GluPro Ala Asp Gly Glu His Leu Val Arg Pro 260 265 270 Leu Arg Glu Leu GlyPro Ile Leu Leu Asp Thr Val Arg Asp Met Pro 275 280 285 Tyr Ala Glu ValGly Thr Ile His His Glu Pro Thr Ser Met Pro Tyr 290 295 300 Val Ala TyrAsp Arg Asn Val Leu Leu Ser Asp Leu Thr Asp Asp Ala 305 310 315 320 ValAsp Ile Ile Val Ala Leu Ala Gly Pro Asp Ala Gly Ala Pro Phe 325 330 335Val Thr Glu Leu Arg His Phe Gly Gly Ala Tyr Ala Arg Pro Pro Lys 340 345350 Val Pro Asn Cys Val Gly Gly Arg Asp Ala Ala Phe Ser Leu Phe Thr 355360 365 Gly Ala Val Pro Glu Ala Glu Gly Leu Arg Arg Arg Asp Asp Leu Leu370 375 380 Asp Arg Leu Arg Pro Trp Ser Thr Gly Gly Thr Asn Leu Asn PheAla 385 390 395 400 Gly Val Glu Asp Ile Ser Pro Ala Ser Val Glu Ala AlaTyr Thr Pro 405 410 415 Ala Asp Phe Ala Arg Leu Arg Ala Val Lys Ala GlnTyr Asp Pro Asp 420 425 430 Asn Met Phe Arg Val Asn Phe Asn Ile Pro ProAla Glu Ser Trp Thr 435 440 445 6 12 PRT Artificial Sequence UNSURE(1)...(2) Unsure 6 Xaa Xaa Thr Gln Trp Gly Trp Ala Leu Glu Xaa Asp 1 510 7 10 PRT Artificial Sequence UNSURE (9)...(9) Unsure 7 Thr Gln TrpGly Trp Ala Leu Glu Xaa Asp 1 5 10 8 21 DNA Streptomyces lavendulae 8cctgttctgg gcggtccgcg g 21 9 24 DNA Streptomyces lavendulae 9 tacgacccggacaacatgtt ccga 24 10 20 DNA Streptomyces lavendulae 10 ccacctcctgctcgtcggcc 20 11 4052 DNA Streptomyces lavendulae 11 acctatccgatgtatgccac cctccacgcc tgccacccgc gcagcctcca gcgcaccctg 60 gcgaagaagggcatccgtcc ggtccacgac gtgtcgatct tctggaccgg gcaggaccgc 120 gacgagctgctgccttccct gctggaggcg gacgtgcagc gcgggcgcgc ggcattggct 180 ctgctggaggagtccgatgt cgtgatcgtc aacctcacga gcatcgaccg ctgttcgcac 240 atctactggcaggagctgga gcacggcccc gagcacgagc ggagagcgcc gtcttcgccg 300 cctaccgcacctgcgaccag gtcatccagg acgccctgcg ggcggccgac gaccgcacca 360 gtgtcgtggccttctcggag ataggcttcg ggccgctgcg caactactgt tccatcaacg 420 acgagatggagcaggcgggt ttcctggcca ccgccgagga cggccgcgtc gagtgggccg 480 gcagcgcggccttcgaggcg gtgcagggca cgcacggggt gaacatcaac ctgcgcgacc 540 gctacaagcacggcctggtc ccggagcgcg actacgagaa ggtccgcacc gacgtcgcgg 600 ccgcctgctggagcggcgca acccccgtac cggcagctgt tcttcgacgc ggtgcgccgc 660 cgggaggaggtctatccggc gaggccaccc agcacgcccc cgacctcatc ctggagccgg 720 cggactggcgctatcttccg ctgggcgacc cgcactgggc ctcgcacgtc caccgcgact 780 ggcactggcgctatcttccg ctgggcgacc cgcactgggc ctcgcacgtc caccgcgact 840 gtgggcgcggcagacccgca ccgccgcccc cgtcgatatt cccgcgaccg tatgcgctct 900 gctcgggcgtgacgtgccga acgactggga cggcgtgccg ctgtcctgaa atcgttgtcc 960 tgtcagcggcgttgactccg gcgggggata ccccgattgg ccaaagtcag cgcgcagtca 1020 ctagcgtacggcgcgtccag cacattcgga cttcgtggtc cggccggccc cggagaattc 1080 agacggcccggcaccggaga ccaatttaaa agtgcaagag aggaacgcgc atgtcagcaa 1140 ggatttccctcttcgcgtgg tggtcgagga catggccaag tcgctggagt tctaccggaa 1200 gctgggcgtcgagatccccg ccgaggccga ctccgcgccg cacacggagg ccgtgctcga 1260 cggcggcatccggctcgcct gggacaccgt ggagacggtg cgcagctacg accccgagtg 1320 gcaggcccccaccggcggcc accgcttcgc catcgcgttc gagttccccg acaccgcgag 1380 cgtggacaagaagtacgccg agctcgtcga cgccggctac gagggccacc tcaagccgtg 1440 gaacgccgtgtggggtcagc gctacgccat cgtcaaggac cccgacggca acgtggtgga 1500 cctcttcgcgcccctcccgt aacaccctgg gcggggcccg gacgcacgcc gcgtcggccg 1560 gtgcgccagctcaccggcac gttccccgaa aggcggacat catggtccta cgagcgcccg 1620 ggccgcgccgcgagccacgt cgcgcgatcg cgccactgcc cgaccgcagc gaacgggaag 1680 aactctgcgggcgggtgaca ttcgcccgcc gggaatacgg cccggccccg gccgatctgc 1740 tcgccgtgcccggttccctg tcggccgccc cgctgggcac cctccgttca gggatccgca 1800 ccggattcctcggcggtccc tggcaggacg gcttcccgcg ctatgtctgg caccggtccg 1860 gtactccgtcgtggaattcc ggctgacggc cgtgcgccgg gcgaatacac cggatacgaa 1920 ctccacccgagcgaatggcc ggaaggggtg gcggaccatg cttcctgagt tccaattgca 1980 gtggaattggctcgacgccc cggccggcgg cggaggcgag ctgcaagcga cctgggcccg 2040 gctgcgcatcgccgtgggcg ccgagaccgt cacactcgtc caggagcccg ggcaggggac 2100 cttccgggagcacacgaccg gctcgctcta ccccctggcc gagtggatcg ccttcaactg 2160 gtggtcgctggtggccgacg cgcggcccgg cacccagata tcccagctgc gcttcgccta 2220 ccgccacggtgtgggcgaca accgcggttc gtggtggatg cgttcgcgcc gtcacatcct 2280 gcgcgccgcctgcgacggct tccgctggcc ggacatgctc ttcgtgcccg agggccggga 2340 gacccggatcgtatggatgc cggacatggg ccccgacgta cgacccggga accgcttcgc 2400 gagccggggcaactcctgtg tggagagcgc cgcgttcacc gccacactgg cctcgttcgt 2460 cgacgcggtgaccgagcgcc tcacggacca gggcatcacc ggcaccccgc tccaggagga 2520 gtgggccgccgtccgcgcca ccgacgagga cgaggccgcc ttctgccgca tcggacggct 2580 gggcctggacccctacgccg aggccgagcc gtacgaggcg gacatgcctc aaggccgccg 2640 agcagttggcggaaccgtcg ccagtgactt cttcaacggg gtgcggcctg agcggatagc 2700 cgaccagctccagtggatcg cgcgcgtccg caccctgatg ggcaccgcgc ccgcggatac 2760 cccgctccctcccgccttgg tggaactgcg caaggactgc gcggacttga gcgagaagtt 2820 cttccgctccggggcgactc gacaacccct gggacctcgg ctacgaggtg cgcaccgggt 2880 gcgcgcgtgggcgggtctgg acgacaccgc gcccttcgac ccggcccccc tgatgggcta 2940 ccgcaccgagcaggtcccct atatggaccg gggcctggtc gccctcggca cccgcagggg 3000 cgcggacgggccggtcctgg tctcctcccg gcgcttcacc gaccgcccgc gccgcttcct 3060 ccaggcccgcgcgctgtggc atctgatctg cgaccccgac gacaccttcc tgattcgcgg 3120 cggcgcacacccaccgccag cacgtggccc gcgcttcgcc ctggaggtcc tggcccccgc 3180 caagggcgtggcgaccctgc tggccgaccc cggacacctg gtgtccgccg aggacgtcga 3240 ggtcatcgccgacgactacg gctgcggcaa catcgtcgtg gaacaccagc tggacaaccg 3300 cgtcctggcgaaggacttca cctggcccgg gccacgcgcc gccggcgcgc cggccggtga 3360 gaggagccggggcgcatgac ctcagccgcc ccgcccgcct ttcccttccc gcccggcccc 3420 ggcggcacggtgccgcccga gtacgcgcgg ctgctcaccg atgacccggt cgccgaggtg 3480 cgcctggcggacggctcgcg catctggctg gtgacccggc acgaggacgt gcgcacggtg 3540 ctcaccgacggccgcttcag ccgccatcgc gccgccatgc tgccgggctc gggcttcggc 3600 cggtcccagggctcgggcat cgtggacctc gacccgccgg agcacggccg gctggcgcgg 3660 tccggtggtggccgcgttcg gtgcctgcgc acggcgcggt tcgcaccccg catcgaggcg 3720 gccgccgaggcggccctgga ccggctgccc gccggcagcg gcacggtgga cctcgtcgcg 3780 gcgtacaccgccccttcgcc ggcccgcgtc acagccgact tcctcgggct gcccggggac 3840 cgtggcaggacgtcacctcc gacgtcgagc tgctgctgct tccgcgcggt gccaccgagc 3900 aggcgctggaaggaggccct gcggcaggct cggccaggtg ctggacgaac tgctcgcggc 3960 ccgaggggccgagccgggcg acagcgtcac cgacacgctg ctggacgcgg aggagctcac 4020 cgacgacgaccggcgcctgc tgctccacgg cc 4052 12 16 PRT Streptomyces lavendulae 12 MetSer Thr Gln Trp Gly Trp Ala Leu Glu Pro Asp Gln Pro Gly Tyr 1 5 10 15 1311 PRT Streptomyces lavendulae 13 Ser Thr Gln Trp Gly Trp Ala Leu GluPro Asp 1 5 10 14 7 PRT Streptomyces lavendulae 14 Leu Phe Trp Ala ValArg Gly 1 5 15 8 PRT Streptomyces lavendulae 15 Tyr Asp Pro Asp Asn MetPhe Arg 1 5 16 12 PRT Streptomyces lavendulae 16 Met Ser Thr Gln Trp GlyTrp Ala Leu Glu Pro Asp 1 5 10 17 105 PRT Streptomyces lavendulae 17 MetThr Ser Ser Asp Gly Ser Asp Leu Thr Thr Leu Val Asn Val Gly 1 5 10 15Arg Ser Val Ala Arg Tyr Phe Glu Arg Ile Gly Ile Thr Glu Ile Ala 20 25 30Gln Leu Arg Asp Arg Asp Pro Val Glu Leu Tyr Glu Arg Met Ser Ala 35 40 45Ala Phe Gly Gln Arg Leu Asp Pro Cys Leu Leu Asp Thr Val Met Ser 50 55 60Ala Val Asp Gln Ala Glu Gly Leu Pro Ala Arg Pro Trp Trp His Tyr 65 70 7580 Thr Pro Glu Arg Lys Arg Leu Leu Ala Gly Glu Gly His Asp Arg Ala 85 9095 Gly Gly Thr Ala Gly Glu Gly Thr Ala 100 105 18 281 PRT Streptomyceslavendulae 18 Met Arg Pro Cys Gly Ser Val Cys Gln His Arg His Phe ArgHis Ala 1 5 10 15 Ala Cys Gly Thr Gln Pro Leu Pro Trp Arg Thr Thr AlaPro Glu Pro 20 25 30 Gly Ala Pro Pro His Glu His Leu Ser Ala Arg Arg LysIle His Ser 35 40 45 Val Ser Pro Glu Tyr Glu Glu Arg Thr Ser Arg Leu ProGly Ala Ile 50 55 60 Gly Trp Val Gln Gly Ala Pro Gly Cys Thr Gly Gly GlyGlu Thr Thr 65 70 75 80 Thr Val Leu Pro Asp Gly Cys Ile Asp Leu Leu TrpThr Ala Gly Arg 85 90 95 Leu Leu Val Ser Gly Pro Asp Thr Ser Ala Tyr SerThr His Arg Gly 100 105 110 Phe Trp Val Gly Val Arg Phe Ser Pro Gly IleAla Pro Ala Leu Leu 115 120 125 Gly Ile Pro Ala His Glu Leu Arg Asp GlnArg Val Asp Leu Ala Asp 130 135 140 Leu Trp Pro Gly Ala Gly Thr Arg ArgLeu Thr Glu Arg Val Asp Gly 145 150 155 160 Gly Gly Arg Thr Arg Pro ProPro Ser Arg Ile Trp His Cys Gly Val 165 170 175 Ala Ala Asp Ala Glu ProVal Asp Pro Leu Leu Arg Ala Val Val Val 180 185 190 Ser Leu Glu Ala GlyArg Ser Val Thr Ala Thr Ala Asp Ser Val Gly 195 200 205 Leu Gly Ala ArgGln Leu His Arg Arg Ser Leu Ala Ala Phe Gly Tyr 210 215 220 Gly Pro LysThr Leu Ala Arg Val Leu Arg Met Gln Arg Ala Leu Arg 225 230 235 240 LeuAla Arg Ala Gly Val Pro Phe Ala Glu Thr Ala Thr Leu Ala Gly 245 250 255Phe Ala Asp Gln Ala His Leu Ala Arg Asp Val Arg Glu Met Ala Gly 260 265270 Ser Ser Leu Ser Glu Leu Val Glu Arg 275 280

What is claimed is:
 1. An isolated expression cassette comprising: apromoter operably linked to a DNA sequence of S. lavendulae whichencodes resistance to mitomycin, wherein the DNA sequence is selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:11.
 2. Anisolated expression cassette comprising: a promoter operably linked to aDNA sequence of S. lavendulae which, when expressed in a cell, confersmitomycin resistance to the cell at greater than 100 mg/ml mitomycin,wherein the DNA sequence is SEQ ID NO:1.
 3. An isolated expressioncassette comprising: a DNA sequence that encodes a polypeptidecomprising SEQ ID NO:5.
 4. An isolated expression cassette comprising: apromoter operably linked to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:11. 5.An isolated expression cassette comprising: a promoter operably linkedto a DNA sequence of S. lavendulae which encodes resistance tomitomycin, wherein the DNA sequence encodes a protein encoded by SEQ IDNO:11.
 6. The expression cassette of claim 1 or 2 wherein SEQ ID NO: 1encodes a polypeptide having a molecular mass of about 56,000 daltons onSodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE). 7.The expression cassette of claim 1, 2, or 3 wherein the promoter isheterologous to S. lavendulae.
 8. A plasmid comprising the expressioncassette of claim
 1. 9. A plasmid comprising the expression cassette ofclaim 2 or
 3. 10. A plasmid comprising the expression cassette of claim4.
 11. A plasmid comprising the expression cassette of claim
 5. 12. Aprobe comprising an isolated nucleic acid sequence comprising at least50 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 11, wherein theprobe detects DNA complementary to DNA encoding mitomycin resistance.13. A probe comprising an isolated nucleic acid sequence comprising atleast 50 contiguous nucleotides of the complement of SEQ ID NO: 1 or SEQID NO: 11, wherein the probe detects DNA encoding mitomycin resistance.14. The probe of claim 12 or 13 wherein the probe is detectably labeled.15. The probe of claim 12 wherein the contiguous nucleotides includenucleotides 310 to 330, nucleotides 1415 to 1438, or nucleotides 641 to661 of SEQ ID NO:
 1. 16. The probe of claim 13 wherein the contiguousnucleotides include the complement of nucleotides 310 to 330,nucleotides 1415 to 1438, or nucleotides 641 to 661 of SEQ ID NO:
 1. 17.The probe of claim 12 wherein the nucleic acid sequence is SEQ ID NO:1.18. The probe of claim 13 wherein the nucleic acid sequence is thecomplement of SEQ ID NO:1.
 19. An isolated transformed cell comprising:a heterologous nucleic acid Sequence comprising a promoter functional inthe cell operably linked to a DNA sequence of S. lavendulae whichencodes resistance to mitomycin, wherein the DNA sequence is selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 11. 20. Anisolated transformed cell comprising: a heterologous nucleic acidsequence comprising a promoter functional in the cell operably linked toa DNA sequence of S. lavendulae which encodes resistance to mitomycin,wherein the DNA sequence is SEQ ID NO:
 11. 21. An isolated transformedcell comprising: a heterologous nucleic acid Sequence comprising apromoter functional in the cell operably linked to a DNA sequence of S.lavendulae which encodes resistance to mitomycin, wherein the DNAsequence encodes SEQ ID NO:
 5. 22. An isolated transformed cellcomprising: a heterologous nucleic acid sequence comprising a promoterfunctional in the cell operably linked to a DNA sequence of S.lavendulae which encodes resistance to mitomycin, wherein the DNAsequence encodes a protein encoded by SEQ ID NO:11.
 23. An isolatedtransformed cell comprising: a heterologous nucleic acid Sequencecomprising a promoter functional in the cell operably linked to a DNAsequence of S. lavendulae, which confers mitomycin resistance to thecell, at greater than 100 mg/ml mitomycin, wherein the DNA sequence isSEQ ID NO:
 1. 24. The isolated transformed cell of claim 19, 20, 21, 22or 23 which is a tumor cell.